Small molecules targeting repeat r(CGG) sequences

ABSTRACT

The invention provides a series of bioactive small molecules that target expanded r(CGG) repeats, termed r(CGG) exp , that causes Fragile X-associated Tremor Ataxia Syndrome (FXTAS). The compound was identified by using information on the chemotypes and RNA motifs that interact. Specifically, 9-hydroxy-5,11-dimethyl-2-(2-(piperidin-1-yl)ethyl)-6H-pyrido[4,3-b]carbazol-2-ium, binds the 5′C G G/3′G G C motifs in r(CGG) exp  and disrupts a toxic r(CGG) exp -protein complex. Specifically, dimeric compounds incorporating two 9-hydroxyellipticine analog structures can even more potently bind the 5′C G G/3′G G C motifs in r(CGG) exp  and disrupts a toxic r(CGG) exp -protein complex. Structure-activity relationships (SAR) studies determined that the alkylated pyridyl and phenolic side chains are important chemotypes that drive molecular recognition of r(CGG) repeats, such as r(CGG) exp . Importantly, the compound is efficacious in FXTAS model cellular systems as evidenced by its ability to improve FXTAS-associated pre-mRNA splicing defects and to reduce the size and number of r(CGG) exp -protein aggregates.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of U.S. provisional applicationSer. No. 61/694,977, filed Aug. 30, 2012, the disclosure of which isincorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbers3R01GM079235-02S1 and 1R01GM079235-01A2, awarded by the NationalInstitutes of Health. The U.S. government has certain rights in theinvention.

BACKGROUND

The development of small molecule chemical probes or therapeutics thattarget RNA remains a significant challenge despite the great interest insuch compounds. The most significant barrier to compound development isa lack of knowledge of the chemical and RNA motif spaces that interactspecifically.

RNA plays diverse and important roles in biological processes (1).Aberrant RNA function causes many severe diseases (2). For example,microRNA disregulation can contribute to cancer (3) and singlenucleotide mutations in mRNAs cause beta-thalassemia and inheritedbreast cancer (4). RNA trinucleotide repeat expansions (termedr(NNN)^(exp) where each rN signifies a ribonucleotide of the repeatedsequence) cause various neurological disorders (5) including Fragile XSyndrome (FXS), Fragile X-associated Tremor Ataxia Syndrome (FXTAS),myolonic dystrophy type 1 (DM1), and Huntington's disease (HD).

Although RNA transcripts with expanded repeats cause the diseasesmentioned above, the physiological response to the repeats and thus thecauses of disease are quite different. Differences are mainly due to thelocation of the expanded repeats in a given mRNA. For example, HD iscaused by an expansion of r(CAG) in the coding region of huntingtinmRNA. In the most well established mechanism of HD, disease is causedwhen expanded r(CAG) repeats are translated into a toxic polyQ versionof huntingtin (6). Thus, HD is caused by a gain-of-function at theprotein level. In FXS, >200 copies of r(CGG) in the 5′ untranslatedregion (UTR) of the fragile X mental retardation 1 (FMR1) mRNA causesdisease by recruiting the ‘RNA-induced initiator of transcriptional genesilencing’ (RITS) complex. The RITS complex then recruits DNAmethyltransferase(s) (DMTases) and/or histone methyltransferases (HMT)to initiate local methylation of the FMR1 gene, causing transcriptionalsilencing (7). Thus, FXS is caused by a loss-of-function. Lastly, FXTASand DM1 are caused when expanded repeats present in UTR's sequesterproteins that are involved in pre-mRNA splicing regulation (8, 9).Sequestration of these proteins causes the aberrant splicing of avariety of pre-mRNAs, leading to the expression of defective proteins.Thus, FXTAS and DM1 are caused by an RNA gain-of-function.

FXTAS is a late onset (over age 50) neurological condition that affectsbalance, tremor, and memory. It affects 1 in 3000 men and 1 in 5000women. FXTAS is caused by expanded CGG-repeat (55-200) alleles in the 5′untranslated region (UTR) of the fragile X mental retardation 1 (FMR1)gene located on the X chromosome. Gain-of-function of r(CGG)^(exp) is ageneral pathogenic mechanism of FXTAS similar to myotonic dystrophy.Evidence for RNA gain-of-function comes from animal models andcell-based assays. For example, insertion of untranslated r(CGG)^(exp)of the length that cause FXTAS into mice and Drosophila causedeleterious effects like those observed in humans that have FXTAS. Incell-based models, r(CGG)^(exp) form nuclear inclusions, and the size ofinclusions scales with the length of the repeat and the age of deathfrom the disease.

A more detailed mechanism for the RNA gain-of-function has recently beenelucidated from studies of patient-derived tissues and model cell lines.In studies by the Charlet group, it was shown that r(CGG)^(exp) firstrecruits DGCR8 followed by recruitment of the Src-Associated substrateduring mitosis of 68 kDa (Sam68) protein. The RNA-protein complex is ascaffold for the assembly of other proteins such as muscleblind-like 1protein (MBNL1) and heterogeneous nuclear ribonucleoprotein (hnRNP).Sam68 is a nuclear RNA-binding protein involved in alternative splicingregulation, and the sequestration of Sam68, MBNL1, and hnRNP byr(CGG)^(exp) leads to the pre-mRNA splicing defects observed in FXTASpatients (see FIG. 1). Targeting r(CGG)^(exp) to inhibit DGCR8 and Sam68binding is an attractive treatment for FXTAS.

Despite the contribution of expanded RNA repeats to diseases, there arefew compounds that target these RNAs in particular and non-ribosomalRNAs in general. Our group recently reported two approaches to design ofsmall molecules (10, 11) and modularly assembled compounds (12) thatbind RNA and modulate its function in vivo. In particular, we have usedinformation about RNA motif-small molecule interactions (13-5) andchemical similarity searching (16-19) to design bioactive ligands thattarget r(CUG)^(exp) and r(CAG)^(exp), which cause DM1 and HD,respectively (10-12).

SUMMARY

The present invention is directed, in various embodiments, to materialsand methods that can interfere with a binding interaction between apathological form of messenger RNA (mRNA) that incorporated extendedrepeat r(CGG) sequences (termed r(CGG)^(exp)), i.e., ribonucleotidesequences wherein the trinucleotide cytosine-guanine-guanine is presentin multiple adjacent repeats, and one or more protein that binds to, oris sequestered by, this structural motif. Extended ribonucleotide COOsequences are believed to form hairpin loops containing non-Watson-CrickG-G base pairs. Compounds and methods of the invention can be used toblock this binding or sequestration interaction between the r(CGG)^(exp)and proteins, such as proteins that would normally carry out mRNAsplicing of exons to yield mature translatable RNA. This r(CGG)^(exp)motif is associated with the genetic disease Fragile X-associated Tremorand Ataxia Syndrome (FXTAS), and compounds and methods of the inventioncan be used for treatment of this medical condition in patientsafflicted therewith.

In various embodiments, the invention provides a r(CGG) binding compoundof formula (I)

R¹ is H, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R² is H, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R³ and R⁴ are independently H, (C1-C6)alkyl, (C1-C6)haloakyl,(C1-C6)alkoxyalkyl (C1-C6)haloalkoxyalkyl, or (C6-C10)aryl;

R⁵ is (C1-C6)alkyl, (C1-C6)alkoxy(C1-C6)alkyl, (C1-C6)haloalkyl,(C1-C6)haloalkoxy, aryl(C1-C6)alkyl, heterocyclyl(C1-C6)alkyl,heteroaryl(C1-C6)alkyl, or (R⁶)₂N—(C1-C6)alkyl, wherein R⁶ is H or(C1-C6)alkyl;

wherein any alkyl, alkanoyl, alkoxy, aryl, heterocyclyl, or heteroarylcan be substituted with 0-3 J groups, wherein J is any of halo,(C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, hydroxy(C1-C6)alkyl,alkoxy(C1-C6)alkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, cyano, nitro,azido, R₂N, R₂NC(O), R₂NC(O)O, R₂NC(O)NR, (C1-C6)alkenyl,(C1-C6)alkynyl, (C6-C10)aryl, (C6-C10)aryloxy, (C6-C10)aroyl,(C6-C10)aryl(C1-C6)alkyl, (C6-C10)aryl(C1-C6)alkoxy,(C6-C10)aryloxy(C1-C6)alkyl, (C6-C10)aryloxy(C1-C6)alkoxy, (3- to9-membered)heterocyclyl, (3- to 9-membered)heterocyclyl(C1-C6)alkyl, (3-to 9-membered)heterocyclyl(C1-C6)alkoxy, (5- to 10-membered)heteroaryl,(5- to 10-membered)heteroaryl(C1-C6)alkyl, (5- to10-membered)heteroaryl(C1-C6)alkoxy, or (5- to 10-membered)heteroaroyl;

R is independently at each occurrence H, (C1-C6)alkyl, or (C6-C10)aryl,wherein any alkyl or aryl group is substituted with 0-3 J;

provided the compound of formula (I) is not any of

or a pharmaceutically acceptable salt thereof. A compound comprisingonly the single ellipticine scaffold, such as a compound of formula (I),is termed a monomeric compound herein. The monomeric compounds of theinvention can be analogs of 9-hydroxyellipticine. In variousembodiments, a compound of the invention is an analog of9-hydroxyellipticine bearing an N-substituted pyridinium moiety.

In various embodiments, the invention also provides a dimeric r(CGG)binding compound of formula (I)

wherein R¹, R², R³, and R⁴ are as defined for the monomeric compound offormula (I), and wherein L is a linker comprising a polypeptide backbonebonded by two respective nitrogen atoms thereof to a nitrogen atom of arespective 1,2,3-triazole group via a respective (C1-C6)alkylene groupoptionally further comprising a glycyl residue, each respective triazolegroup being bonded via a (C1-C6)alkylene group to the respectivepyridinium nitrogen atom of each ellipticine scaffold; or apharmaceutically acceptable salt thereof; or a pharmaceuticallyacceptable salt thereof.

In various embodiments, the dimeric r(CGG) binding compound of formula(II) can comprise a linker of formula (LI)

wherein n=1, 2, 3, 4, 5, 6, 7, or 8; each independently selected n1=0,1, 2, 3, 4, or 5; and each independently selected n2=1, 2, 3, 4, 5, or6; and wherein a wavy line indicates a position of bonding to therespective pyridinium nitrogen atom of formula (II).

In various embodiments, the invention provides pharmaceuticalcomposition comprising a compound of the invention and apharmaceutically acceptable excipient.

In various embodiments, the invention provides a method of inhibiting amessenger RNA molecule with repeat r(CGG) sequence, for example whereinthe repeat r(CGG) sequence is a r(CGG)^(exp) sequence, from binding to aprotein with a binding affinity for a RNA hairpin loop comprising anon-Watson-Crick G-G nucleotide pair, comprising contacting themessenger RNA molecule having the repeat r(CGG), e.g., an expandedr(CGG) (r(CGG)^(exp)), sequence, and an effective amount orconcentration of a compound of formula (I)

wherein

R¹ is K, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R² is H, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R³ and R⁴ are independently H, (C1-C6)alkyl, (C1-C6)haloalkyl,(C1-C6)alkoxyalkyl (C1-C6)haloalkoxyalkyl, or (C6-C10)aryl;

R⁵ is (C1-C6)alkyl, (C1-C6)alkoxy(C1-C6)alkyl, (C1-C6)haloalkyl,(C1-C6)haloalkoxy, aryl(C1-C6)alkyl, heterocyclyl(C1-C6)alkyl,heteroaryl(C1-C6)alkyl, or (R⁶)₂N—(C1-C6)alkyl, wherein R⁶ is H or(C1-C6)alkyl;

wherein any alkyl, alkanoyl, alkoxy, aryl, heterocyclyl, or heteroarylcan be substituted with 0-3 J groups, wherein J is any of halo,(C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, hydroxy(C1-C6)alkyl,alkoxy(C1-C6)alkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, cyano, nitro,azido, R₂N, R₃NC(O), R₂NC(O)O, R₂NC(O)NR, (C1-C6)alkenyl,(C1-C6)alkynyl, (C6-C10)aryl, (C6-C10)aryloxy, (C6-C10)aroyl,(C6-C10)aryl(C1-C6)alkyl, (C6-C10)aryl(C1-C6)alkoxy,(C6-C10)aryloxy(C1-C6)alkyl, (C6-C10)aryloxy(C1-C6)alkoxy, (3- to9-membered)heterocyclyl, (3- to 9-membered)heterocyclyl(C1-C6)alkyl, (3-to 9-membered)heterocyclyl(C1-C6)alkoxy, (5- to 10-membered)heteroaryl,(5- to 10-membered)heteroaryl(C1-C6)alkyl, (5- to10-membered)heteroaryl(C1-C6)alkoxy, or (5- to 10-membered)heteroaroyl;

R is independently at each occurrence H, (C1-C6)alkyl, or (C6-C10)aryl,wherein any alkyl or aryl group is substituted with 0-3 J; or,

an effective amount or concentration of a dimeric r(CGG) bindingcompound of formula (I)

wherein R¹, R², R³, and R⁴ are as defined for the monomeric compound offormula (I), and wherein L is a linker comprising apoly(N-methylalanine) peptide backbone bonded by two respective nitrogenatoms thereof to a nitrogen atom of a respective 1,23-triazole group viaa (C1-C6)alkylene group optionally further comprising a glycyl residue,each respective triazole group being bonded via a (C1-C6)alkylene groupto the respective pyridinium nitrogen atom of the respective9-hydroxyellipticine analogous moiety; or a pharmaceutically acceptablesalt thereof; or a pharmaceutically acceptable salt thereof; or aneffective dose of a pharmaceutical composition comprising a compound asdescribed and a pharmaceutically acceptable excipient.

In various embodiments, the invention provides a method of inhibiting amessenger RNA molecule with an repeat r(CGG) sequence, such as anexpanded r(CGG) sequence (termed a r(CGG)^(exp) sequence herein) frombinding to a protein with a binding affinity for a RNA hairpin loopcomprising a non-Watson-Crick O-G nucleotide pair, comprising contactingthe messenger RNA molecule having the repeat r(CGG) sequence, and aneffective amount or concentration of an analog of 9-hydroxyellipticinecomprising an N-substituted pyridinium moiety.

Accordingly, in various embodiments, the invention provides a method oftreatment of Fragile X-associated Tremor Ataxia Syndrome, comprisingadministering to a patient afflicted therewith a therapeuticallyeffective dose of a compound of formula (I)

wherein

R¹ is H, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R² is H, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R³ and R⁴ are independently H, (C1-C6)alkyl, (C1-C6)haloalkyl,(C1-C6)alkoxyalkyl (C1-C6)haloalkoxyalkyl, or (C6-C10)aryl;

R⁵ is (C1-C6)alkyl, (C1-C6)alkoxy(C1-C6)alkyl, (C1-C6)haloalkyl,(C1-C6)haloalkoxy, aryl(C1-C6)alkyl, heterocyclyl(C1-C6)alkyl,heteroaryl(C1-C6)alkyl, or (R⁶)₂N—(C1-C6)alkyl, wherein R6 is H or(C1-C6)alkyl;

wherein any alkyl, alkanoyl, alkoxy, aryl, heterocyclyl, or heteroarylcan be substituted with 0-3 J groups, wherein J is any of halo,(C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, hydroxy(C1-C6)alkyl,alkoxy(C1-C6)alkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, cyano, nitro,azido, R₂N, R₂NC(O), R₂NC(O)O, R₂NC(O)NR, (C1-C6)alkenyl,(C1-C6)alkynyl, (C6-C10)aryl, (C6-C10)aryloxy, (C6-C10)aroyl,(C6-C10)aryl(C1-C6)alkyl, (C6-C10)aryl(C1-C6)alkoxy,(C6-C10)aryloxy(C1-C6)alkyl, (C6-C10)aryloxy(C1-C6)alkoxy, (3- to9-membered)heterocyclyl, (3- to 9-membered)heterocyclyl(C1-C6)alkyl, (3-to 9-membered)heterocyclyl(C1-C6)alkoxy, (5- to 10-membered)heteroaryl,(5- to 10-membered)heteroaryl(C1-C6)alkyl, (5- to10-membered)heteroaryl(C1-C6)alkoxy, or (5- to 10-membered)heteroaroyl;

R is independently at each occurrence H, (C1-C6)alkyl, or (C6-C10)aryl,wherein any alkyl or aryl group is substituted with 0-3 J;

or a pharmaceutically acceptable salt thereof; or,

an effective amount or concentration of a dimeric r(CGG) bindingcompound of formula (B)

wherein R¹, R², R³, and R are as defined for the monomeric compound offormula (I), and wherein L is a linker comprising apoly(N-methylalanine) peptide backbone bonded by two respective nitrogenatoms thereof to a nitrogen atom of a respective 1,2,3-triazole groupvia a (C1-C6)alkylene group optionally further comprising a glycylresidue, each respective triazole group being bonded via a(C1-C6)alkylene group to the respective pyridinium nitrogen atom of therespective 9-hydroxyellipticine analogous moiety; or a pharmaceuticallyacceptable salt thereof;

or an effective dose of a pharmaceutical composition comprising acompound as described and a pharmaceutically acceptable excipient.

In various embodiments, the invention provides a method of treatment ofFragile X-associated Tremor Ataxia Syndrome, comprising administering toa patient afflicted therewith a therapeutically effective dose of ananalog of 9-hydroxyellipticine comprising an N-substituted pyridiniummoiety, or a dimeric derivative of 9-hydroxyellipticine wherein two9-hydroxyellipticine scaffolds are linked via a linker group.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of the pathogenic mechanism in FXTAS.

FIG. 2 shows a schematic strategy in treatment of FXTAS.

FIG. 3 shows a schematic of the protein displacement assay that was usedto identify small molecule inhibitors of the r(CGG)₁₂-DGCR8Δ interactionand to determine their potencies. The r(CGG)₁₂ oligonucleotide islabeled with a 5′-biotin while DGCR8Δ (blue cloud) contains a histidine(His) tag. Left, in the absence of inhibitor, DGCR8Δ binds to r(CGG)₁₂.Binding is quantified by using two antibodies that form a FRET pair—ananti-His antibody labeled with Tb that binds to DGCR8Δ and streptavidinlabeled with XL665 that binds to r(CGG)₁₂. The two fluorophores arewithin close enough proximity to form a FRET pair. Tb is excited at 345nm; the resulting emission (˜545 nm) excites XL665, which emits at 665nm. Right, in the presence of inhibitor, the r(CGG)₁₂-DGCR8Δ interactionis disrupted, and the two fluorophores are not within close enoughproximity to form a FRET pair. Therefore, emission is only observed at545 nm (due to Tb). XL665 emission is not observed.

FIG. 4 shows the structures of the small molecules identified from anRNA-focused library that inhibit the r(CGG)₁₂-DGCR8Δ interaction andderivatives of the most potent monomeric compound (1a). 1b-1f were usedto construct structure-activity relationships and define the activepharmacopbore. Inhibition is markedly decreased for derivatives 1e and1f (Table 1).

FIGS. 5A and 5B show: 5A: Results of competition dialysis experimentsused to assess the specificity of 1a for r(CGG)₁₂; plot of the amount ofligand bound to various RNAs and DGCR8Δ; 5B: the secondary structures oftwo fully paired RNAs used in competition dialysis (SEQ ID NOs: 1 and2).

FIGS. 6A, 6B, and 6C show data indicating the in vivo efficacy of 1aagainst FXTAS as assessed by improvement in pre-mRNA splicing defects.Briefly, COS7 cells were transfected with an SMN2 or Bcl-x mini-gene inthe presence or absence of a mini-gene that express 60 r(CGG) repeats(CGG 60X) FIG. 6A. The cells were then treated with 1a. Total RNA washarvested, and the alternative splicing of the SMN2 exon 7 or Bcl-x exon2 was determined by RT-PCR. FIG. 6B, 1a improves SMN2 pre-mRNA splicingdefects. FIG. 6C, 1a improves Bcl-x pre-mRNA splicing defects.

FIGS. 7A and 7B show data indicating that compound is decreasesr(CGG)^(exp)-protein aggregates as assessed by fluorescence in situhybridization (FISH). Briefly, COS7 cells were co-transfected with aplasmid encoding 60 r(CGG) repeats and a plasmid encoding GFP. Cellswere then treated with 1a and probed with 5′(CCG)₈-Cy3 DNAoligonucleotide probe. Only cells that are GFP positive were analyzedfor the presence of nuclear foci. Top, FIG. 7A, shows confocalmicroscopy images of cells treated with different concentrations of 1a.For all panels: left, GFP fluorescence (indicates transfected cells);middle, Cy3 fluorescence (indicates r(CGG)^(exp)); right: overlay ofGFP, Cy3, and DAPI (indicates nuclei) fluorescence images. Bottom, FIG.7B, shows a plot of the number of r(CGG) aggregates as a function of theconcentration of 1a.

FIGS. 8A and 88B show the IC₅₀ curve for displacement of DGCR8Δ fromr(CGG)12 by compounds FIG. 8A: compound 1a, and FIG. 8B: compound 1b.

FIGS. 9A, 9B, and 9C show results of the Gel Mobility Shift Assays,showing that DGCR8Δ binds to RNAs with different numbers of r(CGG)repeats similarly; FIG. 9A: 12 c(CGG) repeats, FIG. 9B: 24 c(CGG)repeats; FIG. 9C: 60 c(CGG) repeats.

FIGS. 10A, 10B, and 10C show affinities of compounds 1a and 1b for anRNA containing one 5′CGG/3′GGC motif (SEQ ID NO:3). FIG. 10A, the GCinternal loop RNA containing one 5′CGG/3′GGC motif; FIG. 10B: affinityof compound 1a as evidenced by FRET analysis; FIG. 10C: affinity ofcompound 1b as evidenced by FRET analysis.

FIGS. 1A and 11B show: FIG. 11A: a gel electrophoresis autoradiogram,and FIG. 11B: a graphical plot related to the non-effect of compound 1aon splicing of a PLEKHH2 mini-gene.

FIGS. 12A and 12B show: FIG. 12A: a gel electrophoresis autoradiogram,and FIG. 12B: a graphical plot related to the non-effect of compound 1aon splicing of a cTNT mini-gene.

FIGS. 13A and 13B show synthetic schemes for: FIG. 13A: the E-alkynecompound, and FIG. 13B compound 2E-nNMe, as described further in thetext.

FIG. 14 shows a schematic of the alternative pre-mRNA splicing of SMN2minigene.

FIGS. 15A, 15B, and 15C shows evidence showing reduction of pro-mRNAsplicing defects by compound 2E-5NMe. Briefly, COS7 cells wereco-transfected with plasmids expressing an SMN2 alternative splicingreporter and r(CGG)₆₀. The transfection cocktail was removed, and thecells were incubated with fresh medium containing serially dilutedconcentrations of compound for 24 h. FIG. 15A: a graph showing reductionof pre-mRNA splicing defects of SMN2 minigene figure with compound2E-NMe; 15B: In vivo efficacy of 2E-5NMe against FXTAS as assessed byimprovement in cTNT pre-mRNA splicing defects; 15C: In vive efficacy of2E-5NMe against FXTAS as assessed by improvement in SMN2 pre-mRNAsplicing defects.

DETAILED DESCRIPTION Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

The term “about” as used herein, when referring to a numerical value orrange, allows for a degree of variability in the value or range, forexample, within 10%, or within 5% of a stated value or of a stated limitof a range.

All percent compositions are given as weight-percentages, unlessotherwise stated.

All average molecular weights of polymers are weight-average molecularweights, unless otherwise specified.

Aspects of the present disclosure employ, unless otherwise indicated,techniques of chemistry, and the like, which are within the skill of theart. Such techniques are explained fully in the literature. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this disclosure belongs. Although any methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the present disclosure, the preferred methods andmaterials are now described.

As used herein, “individual” (as in the subject of the treatment) or“patient” means both mammals and non-mammals. Mammals include, forexample, humans; non-human primates, e.g. apes and monkeys; andnon-primates, e.g. dogs, cats, cattle, horses, sheep, and goats.Non-mammals include, for example, fish and birds.

The term “disease” or “disorder” or “condition” or “malcondition” areused interchangeably, and are used to refer to diseases or conditionswherein a repeat r(CGG) sequence, such as r(CGG)^(exp), plays a role inthe biochemical mechanisms involved in the disease or malcondition orsymptom(s) thereof such that a therapeutically beneficial effect can beachieved by acting on a repeat r(CGG) sequence such as r(CGG)^(exp).“Acting on” a repeat r(CGG) sequence, or “modulating” a repeat r(CGG)sequence, can include binding to a repeat r(CGG) sequence, and/orinhibiting the bioactivity of a repeat r(CGG) sequence, and/or blockingthe interaction of a repeat r(CGG) sequence with proteins in vivo. Ther(CGG) sequence can be a r(CGG)^(exp) sequence.

The term “r(CGG)^(exp)” as used herein refers to a pathological form ofmessenger RNA (mRNA) that incorporated extended repeats of r(CGG)sequences (termed r(CGG)^(exp)), i.e., ribonucleotide sequences whereinthe trinucleotide cytosine-guanine-guanine is present in multipleadjacent repeats; or to those domains of the messenger RNA comprisingthe extended r(CGG) repeats, depending upon context. The term “r(CGG)”refers to the ribonucleotide cytosine-guanine-guanine, as is found inribonucleic acids (RNA), and a “repeat r(CGG) sequence” is apolyribonucleotide sequence with one or more tandem repeat of the r(CGG)triplet.

The expression “effective amount”, when used to describe therapy to anindividual suffering from a disorder, refers to the amount of a compoundof the invention that is effective to inhibit or otherwise act on arepeat r(CGG) sequence such as r(CGG)^(exp) in the individual's tissueswherein the repeat r(CGG) sequence such as r(CGG)^(exp) involved in thedisorder is active, such as e.g. in binding of translation or splicingrelated proteins, wherein such inhibition or other action occurs to anextent sufficient to produce a beneficial therapeutic effect, e.g., byblocking the effect of that protein-mRNA interaction.

“Substantially” as the term is used herein means completely or almostcompletely; for example, a composition that is “substantially free” of acomponent either has none of the component or contains such a traceamount that any relevant functional property of the composition isunaffected by the presence of the trace amount, or a compound is“substantially pure” is there are only negligible traces of impuritiespresent.

“Treating” or “treatment” within the meaning herein refers to analleviation of symptoms associated with a disorder or disease, orinhibition of further progression or worsening of those symptoms, orprevention or prophylaxis of the disease or disorder, or curing thedisease or disorder. Similarly, as used herein, an “effective amount” ora “therapeutically effective amount” of a compound of the inventionrefers to an amount of the compound that alleviates, in whole or inpart, symptoms associated with the disorder or condition, or halts orslows further progression or worsening of those symptoms, or prevents orprovides prophylaxis for the disorder or condition. In particular, a“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of compounds of the invention areoutweighed by the therapeutically beneficial effects.

Phrases such as “under conditions suitable to provide” or “underconditions sufficient to yield” or the like, in the context of methodsof synthesis, as used herein refers to reaction conditions, such astime, temperature, solvent, reactant concentrations, and the like, thatare within ordinary skill for an experimenter to vary, that provide auseful quantity or yield of a reaction product. It is not necessary thatthe desired reaction product be the only reaction product or that thestarting materials be entirely consumed, provided the desired reactionproduct can be isolated or otherwise further used.

By “chemically feasible” is meant a bonding arrangement or a compoundwhere the generally understood rules of organic structure are notviolated; for example a structure within a definition of a claim thatwould contain in certain situations a pentavalent carbon atom that wouldnot exist in nature would be understood to not be within the claim. Thestructures disclosed herein, in all of their embodiments are intended toinclude only “chemically feasible” structures, and any recitedstructures that are not chemically feasible, for example in a structureshown with variable atoms or groups, are not intended to be disclosed orclaimed herein.

An “analog” of a chemical structure, as the term is used herein, refersto a chemical structure that preserves substantial similarity with theparent structure, although it may not be readily derived syntheticallyfrom the parent structure. A related chemical structure that is readilyderived synthetically from a parent chemical structure is referred to asa “derivative.”

When a substituent is specified to be an atom or atoms of specifiedidentity, “or a bond”, a configuration is referred to when thesubstituent is “a bond” that the groups that are immediately adjacent tothe specified substituent are directly connected to each other in achemically feasible bonding configuration.

All chiral, diastereomeric, racemic forms of a structure are intended,unless a particular stereochemistry or isomeric form is specificallyindicated. In several instances though an individual stereoisomer isdescribed among specifically claimed compounds, the stereochemicaldesignation does not imply that alternate isomeric forms are lesspreferred, undesired, or not claimed. Compounds used in the presentinvention can include enriched or resolved optical isomers at any or allasymmetric atoms as are apparent from the depictions, at any degree ofenrichment. Both racemic and diastereomeric mixtures, as well as theindividual optical isomers can be isolated or synthesized so as to besubstantially free of their enantiomeric or diastereomeric partners, andthese are all within the scope of the invention.

As used herein, the terms “stable compound” and “stable structure” aremeant to indicate a compound that is sufficiently robust to surviveisolation to a useful degree of purity from a reaction mixture, andformulation into an efficacious therapeutic agent. Only stable compoundsare contemplated herein.

A “small molecule” refers to an organic compound, including anorganometallic compound, of a molecular weight less than about 2 kDa,that is not a polynucleotide, a polypeptide, a polysaccharide, or asynthetic polymer composed of a plurality of repeating units.

As to any of the groups described herein, which contain one or moresubstituents, it is understood that such groups do not contain anysubstitution or substitution patterns that are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisdisclosed subject matter include all stereochemical isomers arising fromthe substitution of these compounds.

Alkyl groups include straight chain and branched alkyl groups andcycloalkyl groups having from 1 to about 20 carbon atoms, and typicallyfrom 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms.Examples of straight chain alkyl groups include those with from 1 to 8carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl,n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groupsinclude, but are not limited to, isopropyl, iso-butyl, sec-butyl,t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As usedherein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkylgroups as well as other branched chain forms of alkyl. Representativesubstituted alkyl groups can be substituted one or more times with anyof the groups listed above, for example, amino, hydroxy, cyano, carboxy,nitro, thio, alkoxy, alkynyl, azido, and halogen groups.

Aryl groups are cyclic aromatic hydrocarbons that do not containheteroatoms in the ring. Thus aryl groups include, but are not limitedto, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl,phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl,biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments,aryl groups contain about 6 to about 14 carbons in the ring portions ofthe groups. Aryl groups can be unsubstituted or substituted, as definedabove. Representative substituted aryl groups can be mono-substituted orsubstituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-,or 6-substituted phenyl or 2-8 substituted naphthyl groups, which can besubstituted with carbon or non-carbon groups such as those listed above.Aryl groups can also bear fused rings, such as fused cycloalkyl rings,within the meaning herein. For example, a tetrahydronaphthyl ring is anexample of an aryl group within the meaning herein. Accordingly, an arylring includes, for example, a partially hydrogenated system, which canbe unsubstituted or substituted, and includes one or more aryl ringssubstituted with groups such as alkyl, alkoxyl, cycloalkyl,cycloalkoxyl, cycloalkylalkyl, cycloalkoxyalkyl, and the like, and alsofused with, e.g., a cycloalkyl ring.

Aralkyl or arylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to anaryl group as defined above. Representative aralkyl groups includebenzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groupssuch as 4-ethyl-indanyl. Aralkenyl group are alkenyl groups as definedabove in which a hydrogen or carbon bond of an alkyl group is replacedwith a bond to an aryl group as defined above.

Heterocyclyl groups or the term “heterocyclyl” includes aromatic andnon-aromatic ring compounds containing 3 or more ring members, of which,one or more is a heteroatom such as, but not limited to, N, O, and S.The sulfur S can be in various oxidized forms, such as sulfoxide S(O) orsulfone S(O)₂. Thus a heterocyclyl can be a cycloheteroalkyl or aheteroaryl, or if polycyclic, any combination thereof. In someembodiments, heterocyclyl groups include 3 to about 20 ring members,whereas other such groups have 3 to about 15 ring members.

Heterocyclyl groups can be monocyclic, or polycyclic, such as bicyclic,tricyclic, or higher cyclic forms. A heterocyclyl group designated as aC₂-heterocyclyl can be a 5-ring with two carbon atoms and threeheteroatoms, a 6-ring with two carbon atoms and four heteroatoms and soforth. Likewise a C₄-heterocyclyl can be a 5-ring with one heteroatom, a6-ring with two heteroatoms, and so forth. The number of carbon atomsplus the number of heteroatoms sums up to equal the total number of ringatoms. A heterocyclyl ring can also include one or more double bonds. Aheteroaryl ring is an embodiment of a heterocyclyl group. The phrase“heterocyclyl group” includes fused ring species including thosecomprising fused aromatic and non-aromatic groups. For example, adioxolanyl ring and a benzodioxolanyl ring system (methylenedioxyphenylring system) are both heterocyclyl groups within the meaning herein. Thephrase also includes polycyclic ring systems containing a heteroatomsuch as, but not limited to, quinuclidyl. Heterocyclyl groups can beunsubstituted, or can be substituted as discussed above. Heterocyclylgroups include, but are not limited to, pyrrolidinyl, piperidinyl,piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl,benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl,indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl,benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl,thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl,isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinylgroups. Representative substituted heterocyclyl groups can bemono-substituted or substituted more than once, such as, but not limitedto, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or6-substituted, or disubstituted with groups such as those listed above.

Heteroaryl groups are aromatic ring compounds containing 5 or more ringmembers, of which, one or more is a heteroatom such as, but not limitedto, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12ring members. A heteroaryl group is a variety of a heterocyclyl groupthat possesses an aromatic electronic structure. A heteroaryl groupdesignated as a C₂-heteroaryl can be a 5-ring with two carbon atoms andthree heteroatoms, a 6-ring with two carbon atoms and four heteroatomsand so forth. Likewise a C₄-heteroaryl can be a 5-ring with oneheteroatom, a 6-ring with two heteroatoms, and so forth. The number ofcarbon atoms plus the number of heteroatoms sums up to equal the totalnumber of ring atoms. Heteroaryl groups include, but are not limited to,groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, thiazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, thiophenyl,benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl,benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl,benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl,thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl,isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinylgroups. Heteroaryl groups can be unsubstituted, or can be substitutedwith groups as is discussed above. Representative substituted heteroarylgroups can be substituted one or more times with groups such as thoselisted above.

Heterocyclylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group as defined above is replacedwith a bond to a heterocyclyl group as defined above. Representativeheterocyclyl alkyl groups include, but are not limited to, furan-2-ylmethyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-ylethyl, and indol-2-yl propyl.

Heteroarylalkyl groups are alkyl groups as defined above in which ahydrogen or carbon bond of an alkyl group is replaced with a bond to aheteroaryl group as defined above.

The term “alkoxy” refers to an oxygen atom connected to an alkyl group,including a cycloalkyl group, as are defined above. Examples of linearalkoxy groups include but are not limited to methoxy, ethoxy, propoxy,butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxyinclude but are not limited to isopropoxy, sec-butoxy, tert-butoxy,isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxyinclude but are not limited to cyclopropyloxy, cyclobutyloxy,cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can includeone to about 12-20 carbon atoms bonded to the oxygen atom, and canfurther include double or triple bonds, and can also includeheteroatoms. For example, an allyloxy group is an alkoxy group withinthe meaning herein. A methoxyethoxy group is also an alkoxy group withinthe meaning herein, as is a methylenedioxy group in a context where twoadjacent atoms of a structures are substituted therewith.

The terms “halo” or “halogen” or “halide” by themselves or as part ofanother substituent mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine.

A “haloalkyl” group includes mono-halo alkyl groups, poly-halo alkylgroups wherein all halo atoms can be the same or different, and per-haloalkyl groups, wherein all hydrogen atoms are replaced by halogen atoms,such as fluoro. Examples of haloalkyl include trifluoromethyl1,1-dichloroethyl, 1,2-dichloroethyl, 1,3-dibromo-3,3-difluoropropyl,perfluorobutyl, and the like.

A “haloalkoxy” group includes mono-halo alkoxy groups, poly-halo alkoxygroups wherein all halo atoms can be the same or different, and per-haloalkoxy groups, wherein all hydrogen atoms are replaced by halogen atoms,such as fluoro. Examples of haloalkoxy include trifluoromethoxy,1,1-dichloroethoxy, 1,2-dichloroethoxy, 1,3-dibromo-3,3-difluoropropoxy,perfluorobutoxy, and the like.

In general, “substituted”, as in a “substituted” group (e.g., alkyl,aryl, etc.) refers to an organic group (alkyl, aryl, etc.) as definedherein in which one or more bonds to a hydrogen atom contained thereinare replaced by one or more bonds to a non-hydrogen atom such as, butnot limited to, a halogen (i.e., F, Cl, Br, and I); an oxygen atom ingroups such as hydroxyl groups, alkoxy groups, aryloxy groups,aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups includingcarboxylic acids, carboxylates, and carboxylate esters; a sulfur atom ingroups such as thiol groups, alkyl and aryl sulfide groups, sulfoxidegroups, sulfone groups, sulfonyl groups, and sulfonamide groups; anitrogen atom in groups such as amines, hydroxylamines, nitriles, nitrogroups, N-oxides, hydrazides, azides, and enamines; and otherheteroatoms in various other groups. Non-limiting examples ofsubstituents that can be bonded to a substituted carbon (or other) atominclude F, Cl, Br, I, OR′, OC(O)N(R′)₂, CN, NO, NO₂, ONO₂, azido, CF₃,OCF, R′, O (oxo), S (thiono), methylenedioxy, ethylenedioxy. N(R′)₂,SR′, SOR′, SO₂R′, SO₂N(R′)₂, SO₃R′, C(O)R′, C(O)C(O)R′, C(O)CH₂C(O)R′,C(S)R′, C(O)OR′, OC(O)R′, C(O)N(R′)₂, OC(O)N(R′)₂, C(S)N(R′)₂,(CH₂)₀₋₂N(R′)C(O)R′, (CH₂)₀₋₂N(R′)N(R′)₂, N(R′)N(R′)C(O)R′,N(R′)N(R′)C(O)OR′, N(R′)N(R′)CON(R′)₂, N(R′)SO₂R′, N(R′)SO₂N(R′)₂,N(R′)C(O)OR′, N(R′)C(O)R′, N(R′)C(S)R′, N(R′)C(O)N(R′)₂,N(R′)C(S)N(R′)₂, N(COR′)CO′, N(OR)′, C(═NH)N(R′)₂, C(O)N(OR′)R′, orC(═NOR′)R′ wherein R′ can be hydrogen or a carbon-based moiety, andwherein the carbon-based moiety can itself be further substituted; forexample, wherein R′ can be hydrogen, alkyl, acyl, cycloalkyl, aryl,aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl, wherein anyalkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, orheteroarylalkyl.

In various embodiments, J is any of halo, (C1-C6)alkyl, (C1-C6)alkoxy,(C1-C6)haloalkyl, hydroxy(C1-C6)alkyl, alkoxy(C1-C6)alkyl,(C1-C6)alkanoyl, (C1-C6)alkanoyloxy, cyano, nitro, azido, R₂N, R₂NC(O),R₂NC(O)O, R₂NC(O)NR, (C1-C6)alkenyl, (C1-C6)alkynyl, (C6-C10)aryl,(C6-C10)aryloxy, (C6-C10)aroyl, (C6-C10)aryl(C1-C6)alkyl,(C6-C10)aryl(C1-C6)alkoxy, (C6-C10)aryloxy(C1-C6)alkyl,(C6-C10)aryloxy(C1-C6)alkoxy, (3- to 9-membered)heterocyclyl, (3- to9-membered)heterocyclyl(C1-C6)alkyl, (3- to9-membered)heterocyclyl(C1-C6)alkoxy, (5- to 10-membered)heteroaryl, (5-to 10-membered)heteroaryl(C1-C6)alkyl, (5- to10-membered)heteroaryl(C1-C6)alkoxy, or (5- to 10-membered)heteroaroyl;wherein R is independently at each occurrence H, (C1-C6)alkyl, or(C6-C10)aryl, wherein any alkyl or aryl group is substituted with 0-3 J.

The term “amino protecting group” or “N-protected” as used herein refersto those groups intended to protect an amino group against undesirablereactions during synthetic procedures and which can later be removed toreveal the amine. Commonly used amino protecting groups are disclosed inProtective Groups in Organic Synthesis, Greene. T. W.; Wuts, P. G. M.,John Wiley & Sons, New York, N.Y., (3rd Edition, 1999). Amino protectinggroups include acyl groups such as formyl, acetyl, propionyl, pivaloyl,t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,trichloroacetyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like; sulfonylgroups such as benzenesulfonyl, p-toluenesulfonyl and the like; alkoxy-or aryloxy-carbonyl groups (which form urethanes with the protectedamine) such as benzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl,p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl,2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl,isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl(Aloc), 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl(Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and thelike; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyland the like; and silyl groups such as trimethylsilyl and the like.Amine protecting groups also include cyclic amino protecting groups suchas phthaloyl and dithiosuccinimidyl, which incorporate the aminonitrogen into a heterocycle. Typically, amino protecting groups includeformyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, phenylsulfonyl, Alloc,Teoc, benzyl, Fmoc, Boc and Cbz. It is well within the skill of theordinary artisan to select and use the appropriate amino protectinggroup for the synthetic task at hand.

The term “hydroxyl protecting group” or “O-protected” as used hereinrefers to those groups intended to protect an OH group againstundesirable reactions during synthetic procedures and which can later beremoved to reveal the amine. Commonly used hydroxyl protecting groupsare disclosed in Protective Groups in Organic Synthesis, Greene, T. W.;Wuts, P. G. M., John Wiley & Sons, New York, N.Y., (3rd Edition, 1999).Hydroxyl protecting groups include acyl groups such as formyl, acetyl,propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl,trifluoroacetyl, trichloroacetyl, o-nitrophenoxyacetyl, α-chlorobutyryl,benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and the like;sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like;acyloxy groups (which form urethanes with the protected amine) such asbenzyloxycarbonyl (Cbz), p-chlorobenzyloxycarbonyl,p-metboxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl,3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl,2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl,2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl,isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl(Alloc), 2,2,2-trichloroethoxycarbonyl, 2-trimethylsilylethyloxycarbonyl(Teoc), phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and thelike; aralkyl groups such as benzyl, triphenylmethyl, benzyloxymethyland the like; and silyl groups such as trimethylsilyl and the like. Itis well within the skill of the ordinary artisan to select and use theappropriate hydroxyl protecting group for the synthetic task at hand.

Standard abbreviations for chemical groups such as are well known in theart can be used herein, and are within ordinary knowledge; e.g.,Me=methyl, Et=ethyl, i-Pr=isopropyl. Bu=butyl, t-Bu=tert-butyl,Ph=phenyl, Bn=benzyl, Ac=acetyl, Bz=benzoyl, and the like.

A “salt” as is well known in the art includes an organic compound suchas a carboxylic acid, a sulfonic acid, or an amine, in ionic form, incombination with a counterion. For example, acids in their anionic formcan form salts with cations such as metal cations, for example sodium,potassium, and the like; with ammonium salts such as NH₄ ⁺ or thecations of various amines, including tetraalkyl ammonium salts such astetramethylammonium, or other cations such as trimethylsulfonium, andthe like. A “pharmaceutically acceptable” or “pharmacologicallyacceptable” salt is a salt formed from an ion that has been approved forhuman consumption and is generally non-toxic, such as a chloride salt ora sodium salt. A “zwitterion” is an internal salt such as can be formedin a molecule that has at least two ionizable groups, one forming ananion and the other a cation, which serve to balance each other. Forexample, amino acids such as glycine can exist in a zwitterionic form. A“zwitterion” is a salt within the meaning herein. The compounds of thepresent invention may take the form of salts. The term “salts” embracesaddition salts of free acids or free bases that are compounds of theinvention. Salts can be “pharmaceutically-acceptable salts.” The term“pharmaceutically-acceptable salt” refers to salts that possess toxicityprofiles within a range that affords utility in pharmaceuticalapplications. Pharmaceutically unacceptable salts may nonethelesspossess properties such as high crystallinity, which have utility in thepractice of the present invention, such as for example utility inprocess of synthesis, purification or formulation of compounds of theinvention.

Suitable pharmaceutically-acceptable acid addition salts may be preparedfrom an inorganic acid or from an organic acid. Examples of inorganicacids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic,sulfuric, and phosphoric acids. Appropriate organic acids may beselected from aliphatic, cycloaliphatic, aromatic, araliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which include formic, acetic, propionic, succinic, glycolic,gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic,fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic,4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic),methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic,trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric,salicylic, galactaric and galacturonic acid. Examples ofpharmaceutically unacceptable acid addition salts include, for example,perchlorates and tetrafluoroborates.

Suitable pharmaceutically acceptable base addition salts of compounds ofthe invention include, for example, metallic salts including alkalimetal, alkaline earth metal and transition metal salts such as, forexample, calcium, magnesium, potassium, sodium and zinc salts.Pharmaceutically acceptable base addition salts also include organicsalts made from basic amines such as, for example,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. Examples ofpharmaceutically unacceptable base addition salts include lithium saltsand cyanate salts. Although pharmaceutically unacceptable salts are notgenerally useful as medicaments, such salts may be useful, for exampleas intermediates in the synthesis of Formula (I) compounds, for examplein their purification by recrystallization. All of these salts may beprepared by conventional means from the corresponding compound accordingto Formula (I) by reacting, for example, the appropriate acid or basewith the compound according to Formula (I). The term “pharmaceuticallyacceptable salts” refers to nontoxic inorganic or organic acid and/orbase addition salts, see, for example, Lit et al., Salt Selection forBasic Drugs (1986), Int J. Pharm., 33, 201-217, incorporated byreference herein.

A “hydrate” is a compound that exists in a composition with watermolecules. The composition can include water in stoichiometricquantities, such as a monohydrate or a dihydrate, or can include waterin random amounts. As the term is used herein a “hydrate” refers to asolid form, i.e., a compound in water solution, while it may behydrated, is not a hydrate as the term is used herein.

A “solvate” is a similar composition except that a solvent other thatwater replaces the water. For example, methanol or ethanol can form an“alcoholate”, which can again be stoichiometric or non-stoichiometric.As the term is used herein a “solvate” refers to a solid form, i.e., acompound in solution in a solvent, while it may be solvated, is not asolvate as the term is used herein.

A “prodrug” as is well known in the art is a substance that can beadministered to a patient where the substance is converted in vivo bythe action of biochemicals within the patient's body, such as enzymes,to the active pharmaceutical ingredient. Examples of prodrugs includeesters of carboxylic acid or carbamic acid groups, which can behydrolyzed by endogenous esterases as are found in the bloodstream ofhumans and other mammals. Endogenous hydrolysis of a carboxylic esterprovides an alcohol and an acid; endogenous hydrolysis of a carbamateyields an alcohol, and amine, and carbon dioxide (throughdecarboxylation of the carbamic acid). Conventional procedures for theselection and preparation of suitable prodrug derivatives are described,for example, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. For example, if X isdescribed as selected from the group consisting of bromine, chlorine,and iodine, claims for X being bromine and claims for X being bromineand chlorine are fully described. Moreover, where features or aspects ofthe invention are described in terms of Markush groups, those skilled inthe art will recognize that the invention is also thereby described interms of any combination of individual members or subgroups of membersof Markush groups. Thus, for example, if X is described as selected fromthe group consisting of bromine, chlorine, and iodine, and Y isdescribed as selected from the group consisting of methyl, ethyl, andpropyl, claims for X being bromine and Y being methyl are fullydescribed.

If a value of a variable that is necessarily an integer, e.g., thenumber of carbon atoms in an alkyl group or the number of substituentson a ring, is described as a range, e.g., 0-4, what is meant is that thevalue can be any integer between 0 and 4 inclusive, i.e., 0, 1, 2, 3, or4.

In various embodiments, the compound or set of compounds, such as areused in the inventive methods, can be any one of any of the combinationsand/or sub-combinations of the listed embodiments.

In various embodiments, a compound as shown in any of the Examples, oramong the exemplary compounds, is provided. Provisos may apply to any ofthe disclosed categories or embodiments wherein any one or more of theother above disclosed embodiments or species may be excluded from suchcategories or embodiments.

The present invention further embraces isolated compounds of theinvention. The expression “isolated compound” refers to a preparation ofa compound of the invention, or a mixture of compounds the invention,wherein the isolated compound has been separated from the reagents used,and/or byproducts formed, in the synthesis of the compound or compounds.“Isolated” does not mean that the preparation is technically pure(homogeneous), but it is sufficiently pure to compound in a form inwhich it can be used therapeutically. Preferably an “isolated compound”refers to a preparation of a compound of the invention or a mixture ofcompounds of the invention, which contains the named compound or mixtureof compounds of the invention in an amount of at least 10 percent byweight of the total weight. Preferably the preparation contains thenamed compound or mixture of compounds in an amount of at least 50percent by weight of the total weight; more preferably at least 80percent by weight of the total weight; and most preferably at least 90percent, at least 95 percent or at least 98 percent by weight of thetotal weight of the preparation.

The compounds of the invention and intermediates may be isolated fromtheir reaction mixtures and purified by standard techniques such asfiltration, liquid-liquid extraction, solid phase extraction,distillation, recrystallization or chromatography, including flashcolumn chromatography, or HPLC.

Description

Overview

All RNA trinucleotide repeats fold into a hairpin with periodicallyrepeating 1×1 nucleotide internal loops in the stem (20). We thereforeprobed an RNA-focused small molecule library enriched with chemotypesthat bind RNA 1×1 nucleotide internal loops, such as the 1×1 nucleotideGG internal loop in r(CGG)^(exp). Since FXTAS is caused by sequestrationof proteins that regulate pre-mRNA splicing, a high throughputprotein-displacement assay was used to screen for inhibitors. From thislibrary, a designer small molecule,9-hydroxy-5,11-dimethyl-2-(2-(piperidin-1-yl)ethyl)-6H-pyrido[4,3-b]carbazol-2-ium,was identified. The compound binds tightly to 1×1 nucleotide GG loopsand is efficacious in cell culture models of FXTAS. Specifically, itimproves pre-mRNA splicing defects and reduces the size and number ofr(CGG)^(exp) nuclear foci. Thus, this compound may serve as a chemicalprobe to understand how r(CGG)^(exp) causes FXS and FXTAS, for whichthere is no treatment. Collectively, these studies suggest that smallmolecules targeting traditionally recalcitrant RNA targets can bedeveloped.

FXTAS is caused by a pathogenic mechanism in which there is again-of-function by an expanded r(CGG) repeat, or r(CGG)^(exp) (20).Like other expanded RNA trinucleotide repeating transcripts,r(CGG)^(exp) folds into a hairpin structure with regularly repeating 1×1nucleotide internal loops, or 5′CGG/3′GGC motifs (FIG. 1) (21). FXTASpatients are carriers of pre-mutation alleles (55-200 repeats) and haveincreased FMR1 mRNA levels and normal or moderately low FMRP proteinexpression levels (22,23). Evidence for RNA gain-of-function comes fromanimal models and cell-based assays. For example, insertion ofuntranslated r(CGG)^(exp) (of the length that cause FXTAS) into mice andDrosophila cause deleterious effects like those observed in humans thathave FXTAS (24, 25). In particular, it has been shown that there isgenetic interaction between r(CGG)^(exp) and Pure mediatesneurodegeneration (26). In cell-based models, r(CGG)^(exp) forms nuclearinclusions, and the size of inclusions scales with the length of therepeat and the age of death from the disease (27, 28).

A more detailed mechanism for the RNA gain-of-function has recently beenelucidated from studies of patient-derived cell lines. In studies by theCharlet group (8), it was shown that r(CGG)^(exp) first recruits DGCR8(29), followed by recruitment of the Src-Associated substrate duringmitosis of 68 kDa protein (Sam68). The RNA-protein complex is a scaffoldfor the assembly of other proteins such as muscleblind-like 1 protein(MBNL1) and heterogeneous nuclear ribonucleoprotein (hnRNP). Sam68 is anuclear RNA-binding protein involved in alternative splicing regulation(30), and the sequestration of Sam68 by r(CGG)^(exp) leads to thepre-mRNA splicing defects observed in FXTAS patients (20). Thus,targeting r(CGG)^(exp) to inhibit protein binding is an attractivetreatment for FXTAS. We therefore screened a library enriched in smallmolecules that are biased, or focused, for binding RNA to identify leadligands that bind r(CGG)^(exp).

In order to construct a library of small molecules that is enriched incompounds that have the potential to recognize RNA 1×1 nucleotideinternal loops like the ones that are displayed in r(CGG)^(exp) (FIG.1), previously reported chemical similarity searches were employed (10,11.). Those searches identified compounds that are similar to thebis-benzimidazole Hoechst 33258, 4′,6-diamidino-2-phenylindole (DAPI),and pentamidine. This RNA-focused collection of small moleculescontained two small molecules that improve defects that are associatedwith r(CAG)^(exp) and r(CUG)^(exp) in cell culture models of HD and DM1,respectively (10, 11). Thus, Hoechst-, pentamidine-, and DAPI-likecompounds were screened to identify inhibitors of the r(CGG)-DGCR8 Δprotein complex.

Screening was completed using a time-resolved FRET assay that has beenpreviously described for identifying inhibitors of ther(CUG)^(exp)-MBNL1 and r(CAG)_(exp)-MBNL1 complexes (FIG. 1) (10, 11).Briefly, a 5′-biotinylated RNA oligonucleotide containing 12 r(CGG)repeats is incubated with Hiss-tagged DGCR8Δ. The ligand of interest isthen added, followed by addition of two antibodies that recognize theRNA (streptavidin-XL665) or DGCR8Δ (Tb labeled anti-His.). If thecompound does not displace DGCR8Δ, then Tb and XL-665 are within closeenough proximity to form a FRET pair. If, however, the ligand displacesthe protein, then no FRET is observed.

From this screen, three compounds (FIG. 2) were identified that disruptthe r(CGG)^(exp)-DGCR8Δ complex in the low to mid micromolar range.(Either no or very slight inhibition was observed for all othercompounds at 100 μM). They include compounds 1a, 2, and Ht-N₃ (FIG. 2).Interestingly, all three compounds were derived from the Hoechst orbis-benzimidazole query. Dose-response curves show that 1a and Ht-Nsdisrupt the r(CGG)₁₂-DGCR8Δ complex with IC₅₀'s of 12 and 33 μM,respectively. Compound 2, however, only disrupts ˜25% of ther(CGG)₁₂-DGCR8Δ□ complex at 100 μM.

Molecular Recognition of r(CGG)^(exp) by 1a.

To further investigate the chemotypes in compound 1a that alloweffective recognition of r(CO)^(exp) and inhibition of ther(CGG)₁₂-DGCR8Δ complex, a series of derivatives were studied (FIG. 2).These compounds probe the role of: (i) the identity of the alkylatedpyridyl side chain; (ii) the phenolic side chain; and, (iii) thepositive charge. The IC₅₀ values for inhibition of protein binding for1b, 1c, and 1d are similar to that of 1a (5-12 μM). The IC₅₀ of compound1e is ˜25 μM while 1f has no effect on protein binding at 25 μM. Table 1summarizes the IC₅₀'s and the percentage of protein displaced fromr(CGG)₁₂ at 25 μM of each compound. Taken together, the presence of apositive charge due to the alkylated pyridyl side chain and the presenceof the exocyclic hydroxyl group are required for compound potency.

In the protein displacement assay, inhibition occurs if the smallmolecule binds the protein or the RNA. Therefore, we used competitiondialysis (31) to assess the selectivity of 1a. A series of RNA targets,including two base paired RNAs, r(CGG)₁₂ (a mimic of r(CGG)^(exp) usedin the displacement assay, FIG. 1), and DGCR8Δ were used (FIG. 3). Theresults of these studies show that 1a binds tightly to r(CGG)₁₂ whilevery little binding is observed to DGCR8Δ. Although some binding isobserved to fully paired RNAs, less than half of the amount of ligandthat partitioned into r(CGG)₁₂ partitioned into these samples. Thus, 1abinds preferentially to r(CGG)₁₂ over the other targets tested. Thebinding affinities of 1a-1d for an RNA with a 1×1 nucleotide GG Internalloop motif were also determined. The measured K_(d)'s are similar forall four compounds and range from ˜40-75 nM (Table 1), as expected basedon their similar potencies.

Biological Activity of 1a in Model Cellular Systems of FXTAS.

In order to assess the bioactivity of 1a, a model cellular system ofFXTAS was used (8). Previously, it has been shown that pre-mRNA splicingdefects are observed in survival of motor neuron 2 (SMN2) and B-celllymphoma x (Bcl-x) mRNAs when cells express r(CGG)^(exp) (8). Thesepre-mRNA splicing defects are due to sequestration of Sam68 byr(CGG)^(exp); Sam68 directly regulates the alternative splicing of SMN2and Bcl-x (a). Specifically, exon 7 of the SMN2 mRNA is included toofrequently in FXTAS model systems; ˜70% of SMN2 mRNA contains exon 7when r(CGG)^(exp) is expressed while exon 7 is included in only ˜30% ofSMN2 mRNA in cells that do not express r(CGG)^(exp) (FIG. 4, top).Likewise, there are two isoforms of Bcl-x mRNA, Bcl-xL and Bcl-xS. InFXTAS cellular model systems, 60% of the Bcl-x mRNA is the Bcl-xLisoform. In healthy cells, only 40% of the mRNA is the Bcl-xL isoform(FIG. 4, bottom).

When cells that express r(CGG)₆₀ are treated with in, improvement inSMN2 and Bcl-x pre-mRNA splicing defects are observed (FIG. 4). Forexample, improvement of SMN2 splicing defects can be observed when cellsare treated with as little as 20 μM of 1a. SMN2 mis-splicing is furtherimproved at higher concentrations: treatment with 100 μM 1a improvespre-mRNA splicing levels to approximately 70% of wild type (absence ofr(CGG)^(exp)) while treatment with 500 μM restores pre-mRNA splicing tolevels wild type (FIG. 4). 1a also improves disregulation of Bcl-xsplicing. Statistically significant improvement is observed when cellsare treated with 100 μM of 1a while restoration of wild type splicingpatterns are observed at 500 μM (FIG. 4). No statistically significanteffect on SMN2 or Bcl-x splicing was observed when cells that do notexpress r(CGG)₆₀ are treated with 1a. This suggests that the improvementof pre-mRNA splicing defects is due to 1a displacing proteins fromr(CGG)₆₀.

Control experiments were also completed to determine the specificity of1a; that is if it affects the splicing of RNAs not controlled by Sam68.In these experiments, a PLEKHH2 (15) or cardiac troponin T (cTNT) (32)mini-gene was used, as their alternative splicing is not regulated bySam68. The addition of 1a (500 μM) did not affect PLEKHH2 or cTNTalternative splicing (Figures S-4 & S-5). Thus, the effect of 1a onpre-mRNA splicing appears to be specific to the splicing of pre-mRNAsregulated by Sam68.

Another phenotype of cells expressing r(CGG)^(exp) is the formation ofnuclear foci. Additional studies were therefore completed by using afluorescence in situ hybridization (FISH) assay to determine if 1adecreased the number or size of foci. As can be observed in FIG. 5, 1areduces the size and the number of foci. Collectively, the improvementin the formation of foci and in the disregulation of pre-mRNAalternative splicing show that 1a binds r(CGG)^(exp) in cellular systemsand displaces bound proteins that are then free to complete their normalphysiological functions.

Comparison to Other Small Molecules that Target RNA.

A few bioactive small molecules have been shown to bind to expandedtriplet repeats in vivo and to improve associated defects (10-12, 15).For example, a bis-benzimidazole (11), pentamidine (15), and modularlyassembled bis-benzimidazoles (12) target the r(CUG)^(exp) that causesDM1. Each improves pre-mRNA splicing defects. In general, modularlyassembled ligands that target multiple 5′CUG/3′GUC motifs inr(CUG)^(exp) simultaneously are the more potent inhibitors. For example,a monomeric bis-benzimidazole (H1) improves pre-mRNA splicing defects inDM1 model systems to wild type levels when 2000 μM of compound is used.A dimeric modularly assembled compound that displays two copies of abis-benzimidazole, 2H-4, improves pre-mRNA splicing levels back to wildtype when cells are treated with a 50 μM solution of the compound. Thisrepresents a greater than 40-fold enhancement in bioactivity provided bya modular assembly approach even though the assembled compounds are ofhigher molecular weight and not classically “drug-like.” The improvedbioactivity of the modularly assembled compound could be due to theincreased surface area occupied by the compound, residence time on theRNA target, and the affinity and selectivity of modularly assembledligands for r(CUG)^(exp) (12, 14).

In order to synthesize second-generation modularly assembled compoundsthat target r(CGG)^(exp), a site that can be used to conjugate 1a-likecompounds onto an assembly scaffold must be identified. Fortuitously,our SAR studies showed that the side chain that emerges from the pyridylgroup can be altered since it does not affect potency. Thus, this siteis ideal for the addition of reactive groups that can be anchored ontoan assembly scaffold.

Implications.

The identification of a bioactive small molecule that targetsr(CGG)^(exp) not only provides lead compounds that could becometherapies for FXTAS, but also other disorders that are mediated byr(CGG)^(exp). Notably, this includes Fragile X Syndrome (FXS), anincurable disease that is the most common single gene cause of autism(33). In this case, FXS is thought to be caused by RNAi-based mechanismin which r(CGG)^(exp) is cleaved into small RNAs that enabletranscriptional silencing (7). Thus, if it is indeed possible toreactivate this locus chemically, then a small molecule that targetsr(CGG)^(exp), and inhibits processing into smaller RNAs could activatethe FMR1 locus.

Lastly, the ability of a small molecule to target r(CGG)^(exp) incellular models of FXTAS and reverse a pre-mRNA splicing defect providesfurther evidence for an RNA gain-of-function mechanism. Since this studyis another example of a small molecule that targets a non-ribosomal RNAthat causes disease, it provides further evidence that small moleculescan be developed to target non-coding regions in RNA even though thesetargets have been thought to be recalcitrant to small moleculeintervention.

Accordingly, the invention provides, in various embodiments, a compoundof formula (I)

wherein

R¹ is H, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R² is H, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R³ and R⁴ are independently H, (C1-C6)alkyl, (C1-C6)haloalkyl,(C1-C6)alkoxyalkyl (C1-C6)haloalkoxyalkyl, or (C6-C10)aryl;

R⁵ is (C1-C6)alkyl, (C1-C6)alkoxy(C1-C6)alkyl, (C1-C6)haloalkyl,(C1-C6)haloalkoxy, aryl(C1-C6)alkyl, heterocyclyl(C1-C6)alkyl,heteroaryl(C1-C6)alkyl, or (R⁶)₂N—(C1-C6)alkyl, wherein R⁶ is H or(C1-C6)alkyl;

wherein any alkyl, alkanoyl, alkoxy, aryl, heterocyclyl, or heteroarylcan be substituted with 0-3 J groups, wherein J is any of halo,(C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, hydroxy(C1-C6)alkyl,alkoxy(C1-C6)alkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, cyano, nitro,azido, R₂N, R₂NC(O), R₂NC(O)O, R₂NC(O)NR, (C1-C6)alkenyl,(C1-C6)alkynyl, (C6-C10)aryl, (C6-C10)aryloxy, (C6-C10)aroyl,(C6-C10)aryl(C1-C6)alkyl, (C6-C10)aryl(C1-C6)alkoxy,(C6-C10)aryloxy(C1-C6)alkyl, (C6-C10)aryloxy(C1-C6)alkoxy, (3- to9-membered)heterocyclyl, (3- to 9-membered)heterocyclyl(C1-C6)alkyl, (3-to 9-membered)heterocyclyl(C1-C6)alkoxy, (5- to 10-membered)heteroaryl,(5- to 10-membered)heteroaryl(C1-C6)alkyl, (5- to10-membered)heteroaryl(C1-C6)alkoxy, or (5- to 10-membered)heteroaroyl;

R is independently at each occurrence H, (C1-C6)alkyl, or (C6-C10)aryl,wherein any alkyl or aryl group is substituted with 0-3 J;

provided the compound of formula (I) is not any of

or a pharmaceutically acceptable salt thereof.

For example, R can be H, or R² can be H, or both can be H.

For example, R³ and R⁴ can each be methyl.

For example, for formula (I) R⁵ can be (R⁶)₂N—(C1-C6)alkyl, wherein R⁶is H or (C1-C6)alkyl; or R⁵ can be (C1-C6)alkyl; or R⁵ can be atriazolylalkyl group, wherein the triazolyl group can be unsubstitutedor can be substituted with 1-3 J groups.

For example, the compound can be an analog of 9-hydroxyellipticinecomprising an N-alkylated pyridinium moiety: formula (I) belowillustrates what is meant by an N-alkylated pyridium moiety, that is,the pyridine ring of the ellipticine is quaternarized by alkylation witha group, shown as R⁵ below, such that the molecule bears a permanentpositive charge. For charge balance, a suitable anion is present, e.g.,halide, sulfate, phosphonate, alkylsulfonate, etc., in the appropriatestoichiometric ratio.

In various embodiments, the invention provides a dimeric r(CGG) bindingcompound that can improve the pre-mRNA defects in FXTAS cellular modelsystems. The dimeric compounds, which comprise two 9-hydroxyellipticineanalogous moieties, can be a dimeric r(CGG) binding compound of formula(II)

wherein R¹, R², R³, and R⁴ are as defined for the monomeric compound offormula (I), and wherein L is a linker comprising a polypeptide backbonebonded by two respective nitrogen atoms thereof to a nitrogen atom of arespective 1, 2, 3-triazole group via a respective (C1-C6)alkylene groupoptionally further comprising a glycyl residue, each respective triazolegroup being bonded via a (C1-C6)alkylene group to the respectivepyridinium nitrogen atom of each ellipticine scaffold; or apharmaceutically acceptable salt thereof; or a pharmaceuticallyacceptable salt thereof.

In various embodiments, the dimeric r(CGG) binding compound of formula(II) can comprise a linker L of formula (LI)

wherein n=1, 2, 3, 4, 5, 6, 7, or 8; each independently selected n1=0,1, 2, 3, 4, or 5; and each independently selected n2=1, 2, 3, 4, 5, or6; and wherein a wavy line indicates a position of bonding to therespective pyridinium nitrogen atom of formula (II).

As the term is used herein, an “ellipticine scaffold” refers to thetetracyclic ring system substituted with groups R¹-R⁴. A “linker” joinsthe two ellipticine scaffolds to form the dimeric r(CGG) bindingcompound of formula (II). The compound of formula (II) can comprise alinker L of formula LI, wherein the two wavy lines indicate points ofbonding to the two pyridinium nitrogen atoms of the two respectiveellipticine scaffolds.

In various embodiments, the invention provides a method of inhibiting amessenger RNA molecule with an repeat r(CGG) sequence, such as anexpanded r(CGG) sequence (a r(CGG)^(exp) sequence) from binding to aprotein with a binding affinity for a RNA hairpin loop comprising anon-Watson-Crick G-O nucleotide pair, comprising contacting themessenger RNA molecule having the repeat r(CGG) sequence, e.g., ar(CGG)^(exp) sequence, and an effective amount or concentration of acompound of formula (I)

wherein

R¹ is H, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R² is H, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R³ and R⁴ are independently H, (C1-C6)alkyl, (C1-C6)haloalkyl,(C1-C6)alkoxyalkyl (C1-C6)haloalkoxyalkyl, or (C6-C10)aryl;

R⁵ is (C1-C6)alkyl, (C1-C6)alkoxy(C1-C6)alkyl, (C1-C6)haloalkyl,(C1-C6)haloalkoxy, aryl(C1-C6)alkyl, heterocyclyl(C1-C6)alkyl,heteroaryl(C1-C6)alkyl, or (R⁶)₂N—(C1-C6)alkyl, wherein R¹ is H or(C1-C6)alkyl;

wherein any alkyl, alkanoyl, alkoxy, aryl, heterocyclyl, or heteroarylcan be substituted with 0-3 J groups, wherein J is any of halo,(C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, hydroxy(C1-C6)alkyl),alkoxy(C1-C6)alkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, cyano, nitro,azido, R₂N, R₂NC(O), R₂NC(O)O, R₂NC(O)NR, (C1-C6)alkenyl,(C1-C6)alkynyl, (C6-C10)aryl, (C6-C10)aryloxy, (C6-C10)aroyl,(C6-C10)aryl(C1-C6)alkyl, (C6-C10)aryl(C1-C6)alkoxy,(C6-C10)aryloxy(C1-C6)alkyl, (C6-C10)aryloxy(C1-C6)alkoxy, (3- to9-membered)heterocyclyl, (3- to 9-membered)heterocyclyl(C1-C6)alkyl, (3-to 9-membered)heterocyclyl(C1-C6)alkoxy, (5- to 10-membered)heteroaryl,(5- to 10-membered)heteroaryl(C1-C6)alkyl, (5- to10-membered)heteroaryl(C1-C6)alkoxy, or (5- to 10-membered)heteroaroyl:

R is independently at each occurrence H, (C1-C6)alkyl, or (C6-C10)aryl,wherein any alkyl or aryl group is substituted with 0-3 J; or,

an effective amount or concentration of a dimeric r(CGG) bindingcompound of formula (II)

wherein R¹, R², R³, and R⁴ are as defined for the monomeric compound offormula (I), and wherein L is a linker comprising a polypeptide backbonebonded by two respective nitrogen atoms thereof to a nitrogen atom of arespective 1,2,3-triazole group via a respective (C1-C6)alkylene groupoptionally further comprising a glycyl residue, each respective triazolegroup being bonded via a (C1-C6)alkylene group to the respectivepyridinium nitrogen atom of each ellipticine scaffold; or apharmaceutically acceptable salt thereof; or a pharmaceuticallyacceptable salt thereof;

or a pharmaceutically acceptable salt thereof.

For example, R¹ can be H, or R² can be H, or both.

For example, R³ and R⁴ can each be methyl.

For example, for formula (I) R³ can be (R⁶)₂N—(C1-C6)alkyl, wherein R⁶is H or (C1-C6)alkyl, or R⁵ can be (C1-C6)alkyl; or R⁵ can be atriazolylalkyl group, wherein the triazolyl group can be unsubstitutedor can be substituted with 1-3 J groups.

More specifically, for practice of the inventive method, the compound offormula (I) can be any of:

or a pharmaceutically acceptable salt thereof.

In various embodiments of the method, the dimeric r(CGG) bindingcompound of formula (II) can comprise a linker of formula (LI)

wherein n=1, 2, 3, 4, 5, 6, 7, or 8; each independently selected n1=0,1, 2, 3, 4, or 5; and each independently selected n2=1, 2, 3, 4, 5, or6; and wherein a wavy line indicates a position of bonding to therespective pyridinium nitrogen atom of formula (II).

For example, the contacting can be in vivo in a patient wherein theinhibiting is medically indicated for treatment of a condition, e.g,wherein the patient is suffering from Fragile X-associated Tremor AtaxiaSyndrome.

In various embodiments, the invention can provide a method of inhibitinga messenger RNA molecule with a repeat r(CGG) sequence, e.g., anexpanded r(CGG) sequence (r(CGG)^(exp)), from binding to a protein witha binding affinity for a RNA hairpin loop comprising a non-Watson-CrickG-G nucleotide pair, comprising contacting the messenger RNA moleculehaving the repeat r(CGG) sequence and an effective amount orconcentration of 9-hydroxyellipticine bearing an N-substitutedpyridinium moiety, or an analog thereof.

The invention provides, in various embodiments, a method of treatment ofFragile X-associated Tremor Ataxia Syndrome, comprising administering toa patient afflicted therewith a therapeutically effective dose of acompound of formula (I)

wherein

R¹ is H, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R² is H, (C1-C6)alkyl, or (C1-C6)alkanoyl;

R³ and R⁴ are independently H, (C1-C6)alkyl, (C1-C6)haloalkyl,(C1-C6)alkoxyalkyl (C1-C6)haloalkoxyalkyl, or (C6-C10)aryl;

R⁵ is (C1-C6)alkyl, (C1-C6)alkoxy(C1-C6)alkyl, (C1-C6)haloalkyl,(C1-C6)haloalkoxy, aryl(C1-C6)alkyl, heterocyclyl(C1-C6)alkyl,heteroaryl(C1-C6)alkyl, or (R⁶)₂N—(C1-C6)alkyl, wherein R¹ is H or(C1-C6)alkyl;

wherein any alkyl, alkanoyl, alkoxy, aryl, heterocyclyl, or heteroarylcan be substituted with 0-3 J groups, wherein J is any of halo,(C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl, hydroxy(C1-C6)alkyl,alkoxy(C1-C6)alkyl, (C1-C6)alkanoyl, (C1-C6)alkanoyloxy, cyano, nitro,azido, R₂N, R₂NC(O). R₂NC(O)O, R₂NC(O)NR, (C1-C6)alkenyl,(C1-C6)alkynyl, (C6-C10)aryl, (C6-C10)aryloxy, (C6-C10)aroyl,(C6-C10)aryl(C1-C6)alkyl, (C6-C10)aryl(C1-C6)alkoxy,(C6-C10)aryloxy(C1-C6)alkyl, (C6-C10)aryloxy(C1-C6)alkoxy, (3- to9-membered)heterocyclyl, (3- to 9-membered)heterocyclyl(C1-C6)alkyl, (3-to 9-membered)heterocyclyl(C1-C6)alkoxy, (5- to 10-membered)heteroaryl,(5- to 10-membered)heteroaryl(C1-C6)alkyl, (5- to10-membered)heteroaryl(C1-C6)alkoxy, or (5- to 10-membered)heteroaroyl;

R is independently at each occurrence H, (C1-C6)alkyl, or (C6-C10)aryl,wherein any alkyl or aryl group is substituted with 0-3 J; or, aneffective amount or concentration of a dimeric r(CGG) binding compoundof formula (II)

wherein R¹, R², R³, and R⁴ are as defined for the monomeric compound offormula (I), and wherein L is a linker comprising a polypeptide backbonebonded by two respective nitrogen atoms thereof to a nitrogen atom of arespective 1,2,3-triazole group via a respective (C1-C6)alkylene groupoptionally further comprising a glycyl residue, each respective triazolegroup being bonded via a (C1-C6)alkylene group to the respectivepyridinium nitrogen atom of each ellipticine scaffold; or apharmaceutically acceptable salt thereof.

For example, R¹ can be H, or R² can be H, or both.

For example, R³ and R⁵ can each be methyl.

For example, for formula (I) R⁵ can be (R⁶)₂N—(C1-C6)alkyl, wherein R⁶is H or (C1-C6)alkyl, or R⁵ can be (C1-C6)alkyl; or R⁵ can be atriazolylalkyl group, wherein the triazolyl group can be unsubstitutedor can be substituted with 1-3 J groups.

More specifically, for practice of the inventive method, the compound offormula (I) can be any of:

or a pharmaceutically acceptable salt thereof.

In various embodiments of the method, the dimeric r(CGG) bindingcompound of formula (II) can comprise a linker of formula (LI)

wherein n=1, 2, 3, 4, 5, 6, 7, or 8; each independently selected n1=0,1, 2, 3, 4, or 5; and each independently selected n2=1, 2, 3, 4, 5, or6; and wherein a wavy line indicates a position of bonding to therespective pyridinium nitrogen atom of formula (II).

In various embodiments, the invention can provide a method of treatmentof Fragile X-associated Tremor Ataxia Syndrome, comprising administeringto a patient afflicted therein, an N-substituted pyridinium moiety, oran analog thereof.

Table 1, below, provides specific data for compounds 1a-1f (see FIG. 4)concerning the potency of the respective compound for disruption of ther(CGG)₁₂-DGCR8Δ complex, expressed as percentage displacement at 25 μM,IC₅₀ (μM) and Kd (nM). The higher the percentage displacement at thefixed concentration of the compound, the more potent the compound is.Compounds 1a-1d were the most potent of the tested compounds.

TABLE 1 The potencies of 1a-1f for disruption of the r(CGG)₁₂-DGCR8Δcomplex and the corresponding affinities for an RNA containing one5′CGG/3′GGC motif. The potencies of the compounds are reported as IC₅₀sas determined from the TR-FRET assay. 1a 1b 1c 1d 1e 1f Percentage 85 ±1  91 ± 5  96 ± 9  87 ± 5   46 ± 5 0 displacement at 25 μM IC₅₀, μM  13± 0.4   8 ± 0.3  13 ± 0.2  7 ± 0.2 ~25 ND^(a) K_(d), nM 76 ± 4  38 ± 1 69 ± 5  50 ± 18  NM^(b) NM^(b) ^(a)ND denotes that no determinationcould be made. ^(b)NM denotes that no measurement was made.

Structure-activity analysis of the monomeric compounds indicatedportions of the 9-hydroxyellipticine analog structure necessary topreserve for RNA sequence binding activity, and portions that could bemodified into a dimeric compound without loss of activity. For example,the oxygen atom on the ellipticine 9-position should be conserved forbioactivity; thus the group designated OR¹ in formula (I) and in formula(II) should be OH or an O-alkyl or O-acyl group. Enhanced bioactivity isobserved when the quaternary, positively-charged pyridinium group at theopposite end of the ellipticine skeleton is also preserved. However, thestructure of the group designated R⁵ in formula (I), i.e., the groupbonded to the quaternary pyridinium nitrogen atom, is not highly relatedto bioactivity. Accordingly, the inventors herein designed a dimericstructure, formula (I), based on these data.

For example, the invention can provide a compound of formula (U) havingas linker L a group of formula (LI), the compound being of formula2E-nNME

with varying numbers of repeat units n for the N-methylalanineoligomeric unit ranging from 1 to about 10 (e.g., whole numbers 1-6,1-8, or 1-10), which is reflected in the nomenclature used herein. Forinstance, when n=1, the compound of formula 2E-nNMe is termed 2E-1NMe,and so forth.

Table 2 presents data indicating the potencies of compound 2E-1 NMethrough 2E-6NMe, i.e., compounds of the above structure with the numberof the N-methylalanine repeating group varying from 1 to 6.

In various embodiments, the invention provides modularly assembled smallmolecules targeting r(CGG)^(exp) that improve the pre-mRNA defects inFXTAS cellular model systems. Modularly assembled compounds display twocopies of an hydroxyl ellipticine-like module that was previously shownto bind r(CGG)^(exp).

The optimal dimeric compounds improve pre-mRNA splicing with 10-foldhigher potency than the monomer, e.g., a compound of formula 1a:

compared to a comparable dimeric compound of formula (II) wherein twoellipticine scaffolds are joined by linker L, e.g., joined by linker(LI).

TABLE 2 Potencies of compounds in inhibition of r(CGG)12-DGCR8Δ complexr(CGG)₁₂-DGCR8Δ complex formation, % 1 μM 10 μM IC₅₀, μM 2E-1NMe 78.0 ±1.2 25.3 ± 2.0 — 2E-2NMe 74.3 ± 2.6 33.0 ± 1.8 — 2E-3NMe 83.4 ± 0.8 17.8± 2.1 — 2E-4NMe 75.6 ± 1.9 18.8 ± 3.7 — 2E-5NMe 39.6 ± 0.6  5.0 ± 2.60.47 ± 0.08 (3.52 ± 0.61)^(a) 2E-6NMe 72.0 ± 0.8  9.2 ± 1.5 — 1a 66.9 ±0.7 20.4 ± 0.8 3.84 ± 0.30 (20.1 ± 1.2)^(a) ^(a)Value in parenthesis isIC₅₀ at the presence of 62 times of competitor, tRNA

Therefore, developing small molecules targeting r(CGG)exp can be astrategy for inhibition of the pathogenic mechanism, proteinsequestration, because small molecules binding to r(CGG)exp can releasethe sequestrated proteins (FIG. 2). However, although attention has beenpaid to targeting RNA for curing RNA-based diseases and considerableeffort has been made to identify small molecules that can interact withRNA, it is challenging to develop bioactive small molecules because of apoor understanding of RNA recognition principles. Previously, weidentified hydroxyellipticine derivatives, such as 1a, as compounds thatcan displace proteins from r(CGG)^(exp) with low micromolar IC₅₀s andbind to r(CGG)^(exp) with nanomolar K_(d)s (FIG. 4 and Table 1). InFXTAS models, the reduced formation of r(CGG)^(exp) aggregation in thepresence of 1a indicates that 1a targets r(CGG)^(exp) in cell cultureand displaces proteins that bind to r(CGG)^(exp). Further evidence ofinhibition of the protein-r(CGG)^(exp) complex in cells is the observedimprovement of pre-mRNA splicing defects by 1a. Thus, 1a displacesprotein from r(CGG)^(exp), allowing these proteins to control pro-mRNAsplicing.

Modular assembly approach is a powerful method to optimize bioactivity(both binding affinity and selectivity) of small molecules targeting therepeats. To better understand binding of 1a to r(CGG)^(exp), astructure-activity relationship analysis was completed. These studiesare summarized herein, and provide an attachment point to constructimproved compounds targeting r(CGG)^(exp) by using a modular assemblystrategy to provide the dimeric compounds of formula (II) having greaterpotency at binding r(CGG) sequences.

Synthesis of a r(CGG)^(exp)-Binding Compound (Module) Suitable forModular Assembly.

r(CGG)^(exp) folds into a hairpin that displays multiple copies of5′CGG/3′GGC motifs. Thus, by displaying a small molecule that binds thisrepeating motif multiple times on the same backbone, high affinity, andselective compounds can be designed to target r(CGG)^(exp). Wepreviously identified that 1a binds 5′CGG/3′GGC motifs and improvesr(CGG)^(exp)-associated defects in cellular models. We thereforesynthesized a derivative that contains an alkyne functional group(9-hydroxy-N-propargylellipticine; E-alkyne; FIG. 13A), such that it canbe conjugated to an azide-functionalized polymeric (N-methyl peptide)backbone (FIG. 13B).

The scheme for the synthesis of E-alkyne is shown in FIG. 13.Ellipticine was synthesized from indole via six reaction steps asreported herein, followed by formylation and Baeyer-Villiger oxidationreaction to obtain 9-hydroxyellipticine. The reaction of9-hydroxyellipticine with propargyl bromide yielded the desired modulecompound containing a reactive ethynyl group, termed, E-alkyne.

Synthesis of N-Methylalanine Peptide Backbone and Modularly AssembledSmall Molecules Targeting r(CGG)^(exp).

In order to display multiple E-alkyne modules on a polymeric backbone, aN-methyl peptide scaffold was employed. Their synthesis is modular innature, allowing for precise control of the valency of ther(CGG)^(exp)-binding modules and the distance between them. That is, thesynthesis of PTAs is iterative: 3-azidopropylamine is coupled to thegrowing PTA backbone (used to couple E-alkyne; controls valency)followed by N-methyl-L-alanine (from 1-6 couplings; controls thedistance between azides) and then 3-azidopropylamine (FIG. 13B). (Thisprocess can be repeated to afford compounds with higher valencies suchas trimers, tetramers, etc.) Following the synthesis of the PTAbackbone, E-alkyne is coupled via a Cu-catalyzed click reaction (FIG.13B). Another advantage of PTAs is that they are more rigid than otherscaffolds such as peptoids, potentially pre-organizing the RNA-bindingmodule for binding r(CGG)^(exp). A library of dimeric compounds(displaying two E-alkyne modules: 2E-nNMe where n denotes the number ofspacing modules) was synthesized by using this approach. After peptidecleavage from Rink amide resin and HPLC purification, the PTAs wereconjugated with E-alkyne and then purified to homogeneity by HPLC.

Screening Dimeric Compounds for Inhibition of r(CGG)^(exp)-DGCR8 Complex

We therefore employed a previously described time-resolved fluorescenceresonance energy transfer (TR-FRET) assay to determine if dimericcompounds can inhibit formation of a r(CGG)₁₂-DGCR8D complex and whichis the most potent. We screened the library of dimers, 2E-1NMe-2E4NMe atboth 1 mM and 10 mM (Table 2). At both concentrations, 2E-5NMe (a dimerwith five N-methyl-L-alanine spacers separating E binding modules) isthe most potent compound: at 1 mM, 60% of the r(CGG)₁₂-DGCR8D complexwas inhibited while at 10 mM, 95% of the complex is inhibited. 2E-5NMehas IC₅₀ of 0.47 mM (Table 2) and is ˜8-fold more potent than themonomer, 1a (Table 1).

Improvement of Pre-mRNA Splicing Defects.

Encouraged by our TR-FRET assay results, we tested the bioactivity ofthe optimal dimeric small molecule, 2E-5NMe, by using a FXTAS cellularmodel. In the FXTAS cell model, r(CGG)^(exp) binds and sequesters Sam68,a pre-mRNA splicing regulator. Sequestration of Sam68 causes itsinactivation and dysregulation of alternative pre-mRNA splicing. Inparticular, the alternative splicing of exon 7 of the survival motorneuron-2 pre-mRNA (SMN2; involved in maintenance of motor neurons andmRNA processing) is dysregulated. Exon 7 is included too frequently whenr(CGG)^(exp) is expressed, ˜70% of time as compared to normal cells inwhich exon 7 has an inclusion rate of only ˜40% (FIG. 14). Experimentswere completed as previously described. Briefly. COS7 cells wereco-transfected with plasmids expressing an SMN2 alternative splicingreporter and r(CGG)₆₀. The transfection cocktail was removed, and thecells were incubated with fresh medium containing serially dilutedconcentrations of compound for 24 h. See FIG. 15A. Total RNA washarvested, and splicing patterns were analyzed via RT-PCR. Remarkably,at 50 mM concentration, 2E-5NMe restores alternative pre-mRNA splicingpatterns back to the wild type. The monomer 1a required 10 times higherconcentration for the same effect. Importantly, 2E-5NMe does not affectalternative splicing in healthy cells or the alternative splicing ofpre-mRNAs not regulated by Sam68 (cardiac troponin T (cTNT) FIG. 15B,and pleckstrin homology domain containing, family H (with MyTH4 domain)member 2 (PLEKHH2), FIG. 15C). These results suggest that theimprovement of the SMN2 pre-mRNA splicing is due to 2E-5NMe bindingr(CGG)₆₀ and displacing proteins from it.

EXAMPLES

Oligonucleotide Preparation and Purification.

The RNAs used in the protein displacement assay (5′-biotin-(CGG)₁₂; SEQID NO:4) and competition dialysis were purchased from Dharmacon. The ACEprotecting groups were cleaved using Dharmacon's deprotection buffer(100 mM acetic acid, adjusted to pH 3.8 with TEMED) by incubating at 60°C. for 2 h. The samples were lyophilized, resuspended in water, anddesalted using a PD-10 gel filtration column (GE Healthcare). Theconcentrations were determined by absorbance at 90° C. using a BeckmanCoulter DU800 UV-Vis spectrophotometer equipped with a Peltiertemperature controller unit. Extinction coefficients (at 260 nm) werecalculated using the HyTher server (34, 35), which uses nearestneighbors parameters (36).

DGCR8Δ Expression and Purification.

His-tagged DGCR8Δ was expressed in Escherichia coli BL21 cells viainduction with 1 mM IPTG for 4 h. Cells were lysed in 50 mL of LysisBuffer (50 mM Tris-Cl pH 8.0, 150 mM NaCl, 2 mM 2-mercaptoethanol, 10 mMimidazole, 0.1% Igepal, 2 mg/mL lysozyme, and 1 mM PMSF) for 30 min onice. DNase I was then added to a final concentration of 1 U/mL, andcells were sonicated (60% power for 9×10 s). The DGCR8Δ protein waspurified via FPLC (Akta Explore, GE Healthcare) using a HiTrap Ni-column(GE Healthcare), followed by a cation exchange column (HiTrap SP FF, GEHealthcare) and a Superdex 75 size exclusion column. The protein wasconcentrated and dialyzed in a Vivaspin 15 centrifugal concentrator(Sartorius Stedim Biotech) into Storage Buffer (10 mM Tris-Cl pH 7.6,200 mM NaCl, 1 mM EDTA, and 5 mM DTI, and 30% Glycerol) and stored at−20° C.

Determination of Compound Potency via a Protein Displacement Assay.

The protein displacement assay used to identify inhibitors of ther(CGG)₁₂-DGCR8Δ complex is based on PubChem BioAssay AID 2675 (FIG. 3),which utilizes time resolved (TR)-FRET between antibodies that bind theRNA and the protein. The assay was conducted in IX TR-PRET Assay Buffer(20 mM HEPES pH 7.5, 110 mM KCl, 110 mM NaCl, 0.1% BSA, 2 mM MgCl₂, 2 mMCaCl₂, 0.05% Tween-20, and 5 mM DTT) with 5 μM yeast extract bulk tRNA(Roche Diagnostics), 160 nM RNA, 154.5 nM His-tagged DOCR8Δ, 40 nMStreptavidin-XL665 (HTRF, Cisbio Bioassays) and 4.4 ng/μl Anti-His₆-Tb(HTRF, Cisbio Bioassays).

The RNA was folded by incubation at 60° C. for 5 min in IX FoldingBuffer (20 mM HEPES, pH 7.5, 110 mM KCl, and 110 mM NaCl) followed byslow cooling to room temperature. Then, DGCR8Δ and the other buffercomponents specified above were added to the folded RNA. Afterincubating for 15 min at room temperature, 9 μL of the mixture wastransferred to a microcentrifuge tube containing 1 μL ligand at varyingconcentrations. A 9 μL aliquot of this final mixture was transferred toa well of a 384-well white plate (Greiner) and incubated for 1 h at roomtemperature. To exclude ligands that perturb F545/F665, a 9 μL controlsolution containing antibodies and different ligand concentrations in1×TR-FRET Assay Buffer but no RNA or protein was also transferred to theplate.

The time resolved fluorescence at 545 nm and 665 nm was measured using aSpectraMax MS plate reader (Molecular Devices, Inc.) with excitationwavelength of 345 nm, cut-off at 420 ran, 200 μs delay, and 1500 μsintegration time. The ratio of fluorescence intensities at 545 nm and665 nm (F545/F665) for a series of ligand dilutions were fit to equation1:

$\begin{matrix}{y = {B + \frac{A - B}{1 + ( \frac{{IC}_{50}}{x} )^{hillslope}}}} & ( {{eq}.\mspace{14mu} 1} )\end{matrix}$where y is the percentage of DGCR8Δ displacement, B is the percentage ofDOCR8Δ displacement in the absence of ligand (0%), A is the maximumpercentage displacement of DGCR8Δ (typically 100%), and the IC₅₀ is theconcentration of ligand where half of the protein is displaced from theRNA. For data from compounds 1a and 1b, see FIG. 8.Competition Dialysis.

Competition dialysis was completed as previously described (31).Briefly, 2 μM RNA or protein was transferred into Slide-a-Lyzer MINIdialysis units with a molecular weight cut-off of 2,000 (ThermoScientific), and the units were placed into a solution of 0.7 μM ligand.Two blank units containing only buffer were used to monitorequilibration by checking the absorbance at the peak wavelength. Afterthe blank units reached equilibrium, sodium dodecyl sulfate (SDS) wasadded to a final concentration of 1%, and the absorbance was measured.This absorbance was used to determine total ligand concentration(C_(t)). The concentration of the dialysate (free ligand concentration.C_(f)) was determined analogously. The bound ligand concentration(C_(b)) was then determined using equation 2:C _(b) =C _(t) −C _(f)  (eq. 2)where C_(b), C_(t), and C_(f) are concentrations of bound, total, andfree ligand, respectively.RNA-Binding Assays Via Dye Displacement.

Dissociation constants were determined using an in-solution,fluorescence-based assay (37-45). RNA was annealed in DNA buffer (8 mMNa₂HPO₄, pH7.0, 185 mM NaCl, 0.1 mM EDTA, 40 μg/mL BSA) at 60° C. for 5min and allowed to slowly cool to room temperature. The annealed RNA wasthen titrated into DNA buffer containing 1000 nM Hoechst 33258.Fluorescence signal was recorded using a Bio-Tek FLX-800 plate reader,which was equipped with excitation filter at 360/40 nm and emissionfilter at 460/40 nm. The change in fluorescence intensity as a functionof RNA concentration was fit to the following equation (eq. 3): (37, 46)I=I ₀+0.5Δϵ{([Ht ₀ ]+[RNA] ₀ +K _(t))−(([Ht] ₀ +[RNA] ₀ +K _(t))²−4[Ht]₀ [RNA] ₀)^(0.5)}  (eq. 3)where I is the observed fluorescence intensity, I₀ is the fluorescenceintensity in the absence of RNA, Δϵ is the difference between thefluorescence intensity in the absence of RNA and in the presence ofinfinite RNA concentration and is in units of M⁻¹. [Ht]₀ is theconcentration of Hoechst 33258, [RNA]₀ is the concentration of theselected internal loop or control RNA, and K_(t) is the dissociationconstant.

Ligands 1a-1d were then added to compete for binding to the RNA (1 μM)in presence of Hoechst 33258 (1 μM). Reduction in fluorescence ofHoechst 33258 was measured using a Bio-Tek FLX-800 plate reader as afunction of ligand concentration (1a-1d) and was fit to the followingequation (eq. 4): (41)

$\begin{matrix}{\theta = {{\frac{1}{{2\lbrack{Ht}\rbrack}_{0}}\begin{bmatrix}{K_{t} + {\frac{K_{t}}{K_{d}}\lbrack C_{t} \rbrack}_{0} + \lbrack{RNA}\rbrack_{0} + \lbrack{Ht}\rbrack_{0} -} \\\sqrt{( {K_{t} + {\frac{K_{t}}{K_{d}}\lbrack C_{t} \rbrack}_{0} + \lbrack{RNA}\rbrack_{0} + \lbrack{Ht}\rbrack_{0}} )^{2} - {{4\lbrack{Ht}\rbrack}_{0}\lbrack{RNA}\rbrack}_{0}}\end{bmatrix}} + A}} & ( {{eq}.\mspace{14mu} 4} )\end{matrix}$where θ is the fraction bound of Hoechst 33258, K_(t) is thedissociation constant for Hoechst 33258, K_(d) is the dissociationconstant of the competing ligand, [Ht]₀ is the total concentration ofthe Hoechst 33258, [C_(t)]₀ is the total concentration of the competingligand, A is the fraction bound of Hoechst 33258 at infiniteconcentration of the competing ligand, and [RNA]₀ is the totalconcentration of RNA. See FIG. 10.Improvement of Splicing Defects in a Cell Culture Model Using RT-PCR.

In order to determine if a improves FXTAS-associated splicing defects invivo, a cell culture model system was used. Briefly. COS7 cells weregrown as monolayers in 24- or 96-well plates in growth medium (1×DMEM,10% FBS, and 1× GlutaMax (Invitrogen)). After the cells reached 90-95%confluency, they were transfected using Lipofectamine 2000 reagent(Invitrogen) or PugenHD (Roche) per the manufacturer's standardprotocol. Equal amounts of a plasmid expressing a 60 CGG repeats and amini-gene of interest (SMN2 or Bcl-x) were used. Approximately 5 hpost-transfection, the transfection cocktail was removed and replacedwith growth medium containing 1a. After 16-24 h, the cells were lysed inthe plate, and total RNA was harvested with a Qiagen RNAEasy kit or aGenElute kit (Sigma). An on-column DNA digestion was completed per themanufacturer's recommended protocol.

A sample of RNA was subjected to reverse transcription-polymerase chainreaction (RT-PCR) using 5 units of AMV Reverse Transcriptase from LifeSciences or Superscript II (Invitrogen). Approximately 300 ng werereverse transcribed, and 150 ng were subjected to PCR. RT-PCR productswere observed after 25-30 cycles of: 95° C. for 1 min: 55° C. for 1 min;72° C. for 2 min and a final extension at 72° C. for 10 min. Theproducts were separated by polyacrylamide or agarose gelelectrophoresis, stained, and imaged using a Typhoon phosphorimager. Thesplicing isoforms were quantified using QuantityOne software (BioRad).Table S-2 lists the RT-PCR primers used for each mini-gene construct.

Two sets of control experiments were completed: (i) COS7 cells wereco-transfected with a control plasmid that does not contain CGG repeatsand the SMN2 or Bcl-x mini-gene as described above; and, (ii) COS7 cellswere co-transfected with the mini-gene that expresses 60 r(CGG) repeatsand a mini-gene that encodes a pre-mRNA whose splicing is not controlledby Sam68 (PLEKHH2 or cTNT) (40. Compound 1a was shown not to effectsplicing of either PLEKHH2 or cTNT. See FIGS. 11 and 12.

SEQ ID NOs: 5-14 are present in the Table below.

TABLE 1 Primer sets used for RT-PCR analysis of alternative splicing.Gene Forward Primer Reverse Primer SMN2 mini-gene 5′GGT GTC CAC TCC CAG TTC AA 5′ GCC TCA CCA CCG TGC TGG Bcl-x mini-gene 5′GGA GCT GGT GGT TGA CTT TCT 5′ TAG AAG GCA CAG TCG AGG cTNT mini-gene 5′GTT CAC AAC CAT CTA AAG CAA 5′ GTT GCA TGG CTG GTG CAG G GAT GPLEKHH2 mini-gene 5′ CGG GGT ACC AAA TGC TGC 5′ CCG CTC GAG CCA TTC ATGAGT TGA CTC TCC AAG TGC ACA GG INSR mini-gene 5′GTA CAA GCT TGA ATG CTG CTC 5′ GCC CTC GAG CGT GGG CAC CTG TCC AAG ACA GGCT GGT CDisruption of Nuclear Foci Using Fluorescence In Situ Hybridization(FISH).

FISH experiments were completed as previously described. (8) Briefly,COS7 cells were plated onto glass coverslips and co-transfected withplasmids encoding for r(CGG)₆₀ and GFP. The cells were fixed in 4%paraformaldehyde in PBS (pH 7.4) for 15 min and washed three times withPBS. Then, they were permeabilized with 0.5% Triton X-100 in PBS. Priorto addition of the FISH probe, the cells were pre-hybridized in a 2×SSCbuffer containing 40% formamide and 10 mg/mL BSA for 30 min. Thecoverslips were hybridized for 2 h in 2×SSC buffer supplemented with 40%formamide, 2 mM vanadyl ribonucleoside, 60 μg/mL tRNA, 30 μg/mL BSA, and0.75 μg (CCG)₈-Cy3 DNA oligonucleotide probe. The cells were washedtwice in 2×SSC containing 50% formamide and then twice in 2×SSC.Following FISH the coverslips were incubated for 10 min in 2×SSCcontaining 1 μg/mL DAPI and rinsed twice in 2×SSC. The coverslips werethen mounted in Pro-Long media and examined using either a simplefluorescence microscope (Leica) or a Leica DM4000 B confocal microscope.

Affinity of DGCR8Δ for Various RNAs Via Gel Mobility Shift Assays

Prior to screening the RNA-focused library for inhibition of ther(CGG)₁₂-DGCR8Δ complex, a gel mobility shift assay was used todetermine the affinity of the protein for various RNAs. Briefly, theRNAs were radioactively labeled by in vitro transcription and[α-32P]-ATP as previously described. (48) The RNAs were folded byincubating the samples at 60° C. in 1× Gel Mobility Shift Buffer (50 mMTris-HCl, pH 8.0, 75 mM NaCl, 37.5 mM KCl, 1 mM MgCl₂, 5.25 mM DTT, and0.1 mg/mL yeast tRNA) excluding the 1 mM MgCl₂ followed by slow coolingon the bench top. Then, MgCl₂ was added to a final concentration of 1 mMand increasing amounts of DGCR8Δ were added to a total volume of 10 ILL.The samples were incubated at mom temperature for 30 min, and then 2 μLof 6× Loading Buffer (40% glycerol, 0.125% Bromophenol Blue, and 0.125%Xylene Cyanol) was added. A 10 μL aliquot of the solution was loaded ona 8% polyacrylamide (80:1 mono/bis) gel pr-chilled in ice water. The gelwas run in 1×TBE for 30 min at 10 V/cm at 0° C., and subsequently driedand exposed to a phosphorimager screen. The gel was imaged using aTyphoon phosphorimager. Protein-RNA binding curves were fit to thefollowing equation:

$y = \frac{{xB}_{\max}}{k_{d} - x}$where y is a percentage of bounded DGCR8Δ, x is the concentration ofprotein. Bmax is maximum percentage of protein bound (restrained toequal 100%), and Kd is disassociation constant, which is approximatelyequal to protein concentration where 50% of maximum binding is achieved.FIG. 9 shows results of the Gel Mobility Shift Assays, showing thatDGCR8Δ binds to RNAs with different numbers of r(CGG) repeats similarly.Kinetic Studies Using Surface Plasmon Resonance.

On rates, off rates and K_(obs) values were measured using a ForteBioOctetRed spectrophotometer and Streptavidin SA dip-and-read biosensors(ForteBio). Sensors were pre-equilibrated in 1× Kinetics Buffer(ForteBio) prior to beginning measurements, 5′-Biotinylated r(CGG)₁₂ wasfolded by heating in IX Kinetics Buffer at 65° C. for 5 min followed byslow cooling to room temperature on the bench top. Measurements werecompleted by incubating sensors sequentially in 200 μL of: 1× KineticsBuffer, 540 nM 5′-biotinylated r(CGG)₁₂, 1× Kinetics Buffer, compound ofinterest (varying concentrations; 1:2 dilutions in 1× Kinetics Buffer),and finally IX Kinetics Buffers. Data were fit using ForteBio's DataAnalysis 7.0 software. Data were fit using a 2:1 heterogeneous ligandmodel. This model fits the binding of one analyte in solution to twodifferent binding sites on the surface. Kinetic parameters arecalculated for both of the interactions.

Small Molecules.

All small molecules 1a-1e were procured from the National CancerInstitute (NCI), Compound 1f, 9-hydroxyellipticine, was obtained fromThe Scripps Research Institute and from VWR, Inc.

Compounds of the invention of formula (I), of compounds that are analogsor derivatives of 9-hydroxyellipticine bearing an N-substitutedpyridinium moiety, can be prepared according to ordinary knowledge inconjunction with the disclosures herein, by a person of ordinary skillin the art of organic synthesis.

The compound 9-hydroxyellipticine is a known compound of formula (A1):

having PubChem Compound ID: 91643; CAS Registry Number 52238-35-4. It iscommercially available, e.g., from Santa Cruz Biotechnology, Inc.,catalog number sc-203940, 2145 Delaware Avenue, Santa Cruz, Calif.95060. U.S.A.

The N-methyl pyridinium analog of ellipticine, also known aselliptinium, as its acetate salt, of formula (A2):

is also a known compound, having Pubchem Compound ID: 42722; CASRegistry Number 58337-35-2.Synthesis of Compounds for Practice of Methods of the Invention

Compounds for practicing methods of the invention include analogs orderivatives of 9-hydroxyelliptcine bearing an N-substituted pyridiniummoiety.

It is within ordinary skill to prepare compounds of this structuralclass or motif from ellipticine, via (1) protection of the phenolichydroxyl group with an O-protecting group, such as are well known in theart; (2) alkylation of the pyridine nitrogen atom with a suitablealkylating agent; then (3) deprotection of the phenolic hydroxyl group.Suitable alkylating agents, such as are well known in the art, caninclude various organic halides, sulfonate esters, and the like. Forexample, reaction of an O)-protected ellipticine with benzyl bromide,followed by O-deprotection, can provide the N-benzyl pyridinium analogof ellipticine, such as can be used in practicing methods of theinvention, or as is a compound of the invention. Similarly, varioussubstituted benzyl bromides can be used to prepare substitutedN-benzylpyridinium analogs of ellipticine, following O-deprotection, asshown in Synthetic Scheme 1, below.

The synthesis of compounds of the invention, or compounds suitable forpracticing methods of the invention, can be prepared according to theabove scheme. The starting material, 9-hydroxyellipticine, is acommercially available compound. In these structures, R³ and R⁴ offormula (I), above, are both methyl, the phenol bears hydrogen (R¹), andthe indole nitrogen bears a hydrogen (R²). First, the phenolic hydroxylgroup of 9-hydroxyellipticine is protected with O-protecting group G,options for which are described in greater detail above, such as arewell-known in the art, to provide the O-protected compound A. Thisintermediate can then be N-alkylated, selectively on the pyridinenitrogen atom, to provide the quaternized pyridinium species B.

As is well-known in the art, the electron-rich pyridine moiety is morereadily alkylated than is the electron-deficient indole moiety.O-deprotection yields the parent compound C, wherein R¹, R² and hydrogenand R³, R⁴ are methyl. R⁵ can be any suitable group, wherein X is aleaving group such as halo, sulfonate ester, and the like; providing areagent useful to alkylate the pyridine nitrogen atom.

For example, R⁵ can be alkyl, or aminoalkyl, or heteroarylalkyl, or thelike. It is within ordinary skill to prepare and use a wide range ofR⁵—X reagents for alkylation of the pyridine nitrogen atom of9-hydroxyellipticine. O-deprotection provides the compound C, whichincludes 9-hydroxyellipticinium compounds of the invention. Furtherreaction of compound C can provide the 9-hydroxyellipticinium compoundsbearing a phenolic ether or ester (i.e., R¹ is alkyl or alkanoyl),compound D, which can be further elaborated under more stringentreaction conditions to provide compounds of the invention in which theindole nitrogen atom bears an alkyl or acyl group (i.e., R² is alkyl oralkanoyl).

For preparation of compounds of the invention wherein R³ and R⁴ areother than the methyl groups found in the ellipticine alkaloids, theperson of ordinary skill can use total synthesis, as shown in SyntheticScheme II, below.

For further details, see: Heterocylic chemistry, 48, 814, (2011); theroute from that product to the product of reaction “h” is described inSynthesis, page 1221 (1992). By use of reagents other thanhexane-2,5-dione in step a, final products with R³ and R⁴ groups otherthan methyl can readily be obtained by the person of ordinary skill;e.g. compounds with hydrogen or with various alkyl, haloalkyl,alkoxyalkyl, haloalkoxyalkyl, and/or aryl groups as groups R³ and R⁴.When a dione other than hexane-2,5-dione is used in Step a of SyntheticScheme II, the indole product of that reaction will comprise analogs ofthe reaction product shown wherein R³ and R⁴ are other than methyl.Carrying this intermediate through to the product of Step h yield ananalog of 9-hydroxyellipticine, wherein R³ and R⁴ are the groupsincorporated in Step a, e.g., other alkyl groups, aryl groups,heteroaryl groups, and the like. This intermediate can be converted intothe N-alkylpyridinium species as indicated in Synthetic Scheme I.

In some instances, using highly reactive alkylating agent such as thepropargyl halide shown in Step i of Synthetic Scheme II, O-protection isnot necessary, as selective N-alkylation can be achieved, for example,to yield the N-propargyl pyridinium product of Step i of SyntheticScheme II.

The reactive triple bond of this propargyl species can be used as aprecursor for further elaboration of heteroaryl-comprising R⁵ groups,such as by the use of click chemistry and the acetylene-azide clickreaction, to yield triazole-alkyl groups at position R⁵. The triazoleitself can bear additional groups, e.g., additional amino groups and thelike, through use of the appropriate azido precursor, as is apparent toa person of skill in the art of organic synthesis.

For synthesis of the dimeric compounds of formula (II), FIG. 13 shows asynthetic scheme that in conjunction with ordinary knowledge of theperson having skill in the art serves to teach how to prepare compoundsof formula (II) of the invention. In FIG. 13, the steps are as follows:Synthetic scheme of E-alkyne: (a) hexane-2,5-dione, p-TsOH, EtOH,reflux; (b) POCl₃, DMF, chlorobenzene, reflux; (c) aminoacetaldehydediethylacetal, 110° C.; (d) NaBH4; (e) p-TsCl, pyridine, rt.; (f) HCl,dioxane, reflux; (g) HMTA/TFA, reflux; (h) H₂SO₄/H₂O₂, MeOH, reflux,52%; (i) 3-byromoprop-1-yne, DMF, 62%. B. synthetic scheme of 2E-nNMe:(j) 20% piperizine/DMF: 2-bromoacetic acid, DIC, DIPEA/DMF, microwave:(k) 3-azidopropylamine/DMF, microwave; (1) Fmoc-N-methyl-L-alanine. DIC,HOAt, DIEA/DMF, microwave at 75° C.; 20% piperizine/DMF; (m)2-bromoacetic acid, DIC, DIPEA/DMF, microwave; 3-azidopropylamine/DMF,microwave; (n) 30% TFA/CH₂Cl₂; HPLC purification; (o) CuSO₄, Naascorbate, TBTA/H₂O:tBuOH=1:1, sonication.

The reactive triple bond of the propargyl species, termed the E-alkyneherein, can undergo the acetylene-azide click reaction, e.g.,copper-catalyzed click reaction, to form the triazole rings of thelinker L of formula LI as described above.

Synthesis.

Fmoc-Rink amide resin (059 mmol/g) was purchased from Advanced ChemTech.N,N-dimethylformamide (DMF, anhydrous) was purchased from EMD and usedwithout further purification. Piperidine, trifluoroacetic acid (TFA),N,N-diisopropylethyl amine (DIEA), and 2-bromoacetic acid were purchasedfrom Sigma Aldrich. N,N′-diisopmpylcarbodiimide (DIC) and1-hydroxy-7-azabenzotriazole (HOAt) were purchased from AdvancedChemTech. Fmoc-N-methyl-L-alanine was purchased from Combi-Blocks,9-Hydroxyellipticine was synthesized as reported previously, (22,23)N-methyl alanine peptides were synthesized using a Biotage Initiator+SP Wave microwave.

Compound Purification and Analysis.

Preparative HPLC was performed using a Waters 1525 Binary HPLC pumpequipped with a Waters 2487 dual absorbance detector system and a WatersSunfire C18 OBD 5 μm 19×150 mm column. Absorbance was monitored at 315and 220 nm. A gradient of 20-100% MeOH in H₂O with 0.1% TFA over 60 minwas used for compound purification. Analytical HPLC was performed usinga Waters Symmetry C18 5 μm 4.6×150 mm column. Compounds were analyzedusing a gradient of 20-100% MeOH in H₂O with 0.1% TFA over 60 min. Allcompounds evaluated had ≥95% purity by analytical HPLC. Massspectrometry was performed with an Applied Biosystems MALDI ToF/ToFAnalyzer 4800 Plus using an α-hydroxycinnamic acid matrix.

Synthesis of N-Methyl-L-Alanine Peptide Backbone.

Deprotected Rink amide resin (200 mg, 0.12 mmol) was shaken with asolution of 1 M bromoacetic acid (2 mL) and DIC (250 μL, 1.5 mmol) inDMF (2 mL) via microwave irradiation (3×15 s) using a 700 W microwaveset to 10% power. The resin was washed with DMF (3×5 mL) and reactedtwice with a solution of 3-azidopropylamine (250 μL, 0.6 mmol) in DMF (2mL) via microwave irradiation (3×15 s) using a 700 W microwave set to10% power. The resin was washed with DMF (3×5 mL). Then a solution ofFmoc-N-methyl-L-alanine (100 mg, 03 mmol), DIC (48 μL, 0.9 mmol), HOAt(41 mg, 0.9 mmoL), and DIEA (104 μL, 0.9 mmol) in DMF (2 mL) was addedand the reaction heated via microwave to 75° C. for 10 min. The resinwas washed with DMF and the FMOC was removed with 20% piperidine/DMF(2×10 min). This cycle was repeated until a desired number ofN-methyl-L-alanine was added. The resin was shaken with a solution of 1M bromoacetic acid (2 mL) and DIC (250 μL, 1.5 mmol) in DMF (2 mL) viamicrowave irradiation (3×15 s) using a 700 W microwave set to 10% power.The resin was washed with DMF (3×5 mL) and reacted twice with a solutionof 3-azidopropylamine (250 μL, 0.6 mmol) in DMF (2 mL) via microwaveirradiation (3×15 s) using a 700 W microwave set to 10% power. Thepeptides were cleaved from the resin by 30% TFA/CH₃Cl₂ and purified byHPLC.

Synthesis of 9-hydroxy-N-propargylellipticine

Into the solution of 9-hydroxyellipticine (100 mg, 038 mmol) in DMF, wasadded propargyl bromide (023 mL, 2.1 mmol) and the solution stirredovernight at room temperature. After diethyl ether was added, theproduct (62%) was obtained by filtration. ¹H-NMR (400 MHz, DMSO-d6) δ2.82 (s, 3Hs), 3.26 (s, 3Hs), 3.98 (s, 1H), 5.69 (s, 2Hs), 7.17 (dd, 1H,1=8 Hz, J=4 Hz), 7.51 (d, 1H, J=8 Hz), 7.81 (d, 1H, J=4 Hz), 8.49 (d,2H, J=4 Hz), 9.43 (s, 1H), 10.12 (s, 1H), 12.03 (a, 1H). ¹³C-NMR (400MHz, DMSO-d6) δ 11.95, 14.84, 48.35, 76.95, 80.12, 109.77, 110, 32,112.22, 117.57, 119.49, 120.55, 122.93, 126.21, 129.88, 132.16, 134.05,136.04, 145.19, 146.50, 152.19.

General procedure for 9-hydroxy-N-propargylellipticine conjugation topeptide tertiary amides

Peptide backbone was dissolved in a 1:1 mixture of tBuOH and H₂O andCuSO₄, sodium ascorbate, TBTA and 9-hydroxy-N-propargylellipticine wereadded in the solution. The mixture was sonicated for 3 hours and theconjugate was purified by using reverse phase HPLC with 20-75%MeOH/H₂O+0.1% (v/v) TFA over 40 min.

Characterization of Compounds for Practice of Methods of the Invention

The purities of the compounds used in additional studies (IC₅₀'s,affinities, etc.) were determined by HPLC, and their masses wereconfirmed by ESI mass spectrometry. All compounds were ≥95% pure. Massspectra were collected on a Varian 500 MS spectrometer equipped withVarian Prostar Autosampler 410. The purities of compounds weredetermined by analytical HPLC using a Waters 1525 Binary HPLC Pumpequipped with Waters 2487 Dual λ Absorbance Detector system and thefollowing conditions: a Waters Symmetry C8 5 μm 4.6×150 mm column, roomtemperature, flow rate 2.4 ml/min, and a linear gradient of 0-100% B inA for 60 min. A is water, B is methanol.

These data are shown below in Table S-2.

TABLE S-2 Characterization of 1a and derivatives thereof including HPLCretention times, and calculated and observed masses. Molecular HPLCRetention MS ES(+)-MS Compound Formula Time (min) (Calculated) (Found)1a C₂₄H₂₈N₃O⁺ 21 374.2 (M) 1b C₂₃H₂₈N₃O⁺ 14 362.2 (M) 362.3 (M) 1cC₁₉H₁₉N₂O⁺ 18 291.2 (M) 291.2 (M) 1d C₁₈H₁₇N₂O⁺ 17 277.1 (M) 277.1 (M)1e C₁₇H₁₄N₂O 29 263.1 263.1 (M + H)⁺ (M + H)⁺ 1f C₁₇H₁₄N₂ 32 247.1 247.1(M + H)⁺ (M + H)⁺Evaluations

It is within ordinary skill using the procedures provided herein and inreferences cited herein, which are incorporated by reference in theirentireties, to evaluate any compound disclosed and claimed herein foreffectiveness for in vivo evaluation of bioactivity ofr(CGG)^(exp)-binding small molecules, as well as in the various cellularassays found in the scientific literature. Accordingly, the person ofordinary skill, using the disclosure of the present application inconjunction with the disclosures of documents cited herein, and theknowledge of the person of ordinary skill, can prepare and evaluate anyof the claimed compounds for effectiveness as a potential humantherapeutic agent, without undue experimentation.

Any r(CGG)^(exp)-binding small molecule compound found to be effectiveas an bioactive agent can likewise be further tested in animal models,and in human clinical studies, using the skill and experience of theinvestigator to guide the selection of dosages and treatment regimens.

Pharmaceutical Compositions of the Invention and for Use in Methods ofthe Invention

Another aspect of an embodiment of the invention provides compositionsof the compounds of the invention, alone or in combination with anothermedicament. As set forth herein, compounds of the invention includestereoisomers, tautomers, solvates, prodrugs, pharmaceuticallyacceptable salts and mixtures thereof. Compositions containing acompound of the invention can be prepared by conventional techniques,e.g. as described in Remington: The Science and Practice of Pharmacy,19th Ed., 1995, or later versions thereof, incorporated by referenceherein. The compositions can appear in conventional forms, for examplecapsules, tablets, aerosols, solutions, suspensions or topicalapplications.

Typical compositions include a compound of the invention and apharmaceutically acceptable excipient that can be a carrier or adiluent. For example, the active compound will usually be mixed with acarrier, or diluted by a carrier, or enclosed within a carrier that canbe in the form of an ampoule, capsule, sachet, paper, or othercontainer. When the active compound is mixed with a carrier, or when thecarrier serves as a diluent, it can be solid, semi-solid, or liquidmaterial that acts as a vehicle, excipient, or medium for the activecompound. The active compound can be adsorbed on a granular solidcarrier, for example contained in a sachet. Some examples of suitablecarriers are water, salt solutions, alcohols, polyethylene glycols,polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin,lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar,cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin,acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid,fatty acids, fatty acid amines, fatty acid monoglycerides anddiglycerides, pentaerythritol fatty acid esters, polyoxyethylene,hydroxymethylcellulose and polyvinylpyrrolidone. Similarly, the carrieror diluent can include any sustained release material known in the art,such as glyceryl monostearate or glyceryl distearate, alone or mixedwith a wax.

The formulations can be mixed with auxiliary agents that do notdeleteriously react with the active compounds. Such additives caninclude wetting agents, emulsifying and suspending agents, salt forinfluencing osmotic pressure, buffers and/or coloring substancespreserving agents, sweetening agents or flavoring agents. Thecompositions can also be sterilized if desired.

The route of administration can be any route which effectivelytransports the active compound of the invention to the appropriate ordesired site of action, such as oral, nasal, pulmonary, buccal,subdermal, intradermal, transdermal or parenteral, e.g., rectal, depot,subcutaneous, intravenous, intraurethral, intramuscular, intranasal,ophthalmic solution or an ointment, the oral route being preferred.

If a solid carrier is used for oral administration, the preparation canbe tableted, placed in a hard gelatin capsule in powder or pellet formor it can be in the form of a troche or lozenge. If a liquid carrier isused, the preparation can be in the form of a syrup, emulsion, softgelatin capsule or sterile injectable liquid such as an aqueous ornon-aqueous liquid suspension or solution.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which can be prepared using a suitable dispersant or wettingagent and a suspending agent. Injectable forms can be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution.Alternatively, sterile oils can be employed as solvents or suspendingagents. Preferably, the oil or fatty acid is non-volatile, includingnatural or synthetic oils, fatty acids, mono-, dl- or tri-glycerides.

For injection, the formulation can also be a powder suitable forreconstitution with an appropriate solution as described above. Examplesof these include, but are not limited to, freeze dried, rotary dried orspray dried powders, amorphous powders, granules, precipitates, orparticulates. For injection, the formulations can optionally containstabilizers, pH modifiers, surfactants, bioavailability modifiers andcombinations of these. The compounds can be formulated for parenteraladministration by injection such as by bolus injection or continuousinfusion. A unit dosage form for injection can be in ampoules or inmulti-dose containers.

The formulations of the invention can be designed to provide quick,sustained, or delayed release of the active ingredient afteradministration to the patient by employing procedures well known in theart. Thus, the formulations can also be formulated for controlledrelease or for slow release.

Compositions contemplated by the present invention can include, forexample, micelles or liposomes, or some other encapsulated form, or canbe administered in an extended release form to provide a prolongedstorage and/or delivery effect. Therefore, the formulations can becompressed into pellets or cylinders and implanted intramuscularly orsubcutaneously as depot injections. Such implants can employ known inertmaterials such as silicones and biodegradable polymers, e.g.,polylactide-polyglycolide. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides).

For nasal administration, the preparation can contain a compound of theinvention, dissolved or suspended in a liquid carrier, preferably anaqueous carrier, for aerosol application. The carrier can containadditives such as solubilizing agents, e.g., propylene glycol,surfactants, absorption enhancers such as lecithin (phosphatidylcholine)or cyclodextrin, or preservatives such as parabens.

For parenteral application, particularly suitable are injectablesolutions or suspensions, preferably aqueous solutions with the activecompound dissolved in polyhydroxylated castor oil.

Tablets, dragees, or capsules having talc and/or a carbohydrate carrieror binder or the like are particularly suitable for oral application.Preferable carriers for tablets, dragees, or capsules include lactose,cornstarch, and/or potato starch. A syrup or elixir can be used in caseswhere a sweetened vehicle can be employed.

A typical tablet that can be prepared by conventional tabletingtechniques can contain:

Core: Active compound (as free compound or salt thereof) 250 mgColloidal silicon dioxide (Aerosil ®) 1.5 mg Cellulose, microcryst.(Avicel ®) 70 mg Modified cellulose gum (Ac-Di-Sol ®) 7.5 mg Magnesiumstearate Ad. Coating: HPMC approx. 9 mg *Mywacett 9-40 T approx. 0.9 mg*Acylated monoglyceride used as plasticizer for film coating.

A typical capsule for oral administration contains compounds of theinvention (250 mg), lactose (75 mg) and magnesium stearate (15 mg). Themixture is passed through a 60 mesh sieve and packed into a No. 1gelatin capsule. A typical injectable preparation is produced byaseptically placing 250 mg of compounds of the invention into a vial,aseptically freeze-drying and sealing. For use, the contents of the vialare mixed with 2 mL of sterile physiological saline, to produce aninjectable preparation.

The compounds of the invention can be administered to a mammal,especially a human in need of such treatment, prevention, elimination,alleviation or amelioration of a malcondition. Such mammals include alsoanimals, both domestic animals, e.g. household pets, farm animals, andnon-domestic animals such as wildlife.

The compounds of the invention are effective over a wide dosage range.For example, in the treatment of adult humans, dosages from about 0.05to about 5000 mg, preferably from about 1 to about 2000 mg, and morepreferably between about 2 and about 2000 mg per day can be used. Atypical dosage is about 10 mg to about 1000 mg per day. In choosing aregimen for patients it can frequently be necessary to begin with ahigher dosage and when the condition is under control to reduce thedosage. The exact dosage will depend upon the activity of the compound,mode of administration, on the therapy desired, form in whichadministered, the subject to be treated and the body weight of thesubject to be treated, and the preference and experience of thephysician or veterinarian in charge.

Generally, the compounds of the invention are dispensed in unit dosageform including from about 0.05 mg to about 1000 mg of active ingredienttogether with a pharmaceutically acceptable carrier per unit dosage.

Usually, dosage forms suitable for oral, nasal, pulmonal or transdermaladministration include from about 125 μg to about 1250 mg, preferablyfrom about 250 μg to about 500 mg, and more preferably from about 2.5 mgto about 250 mg, of the compounds admixed with a pharmaceuticallyacceptable carrier or diluent.

Dosage forms can be administered daily, or more than once a day, such astwice or thrice daily. Alternatively dosage forms can be administeredless frequently than daily, such as every other day, or weekly, if foundto be advisable by a prescribing physician.

DOCUMENTS CITED

-   1. Atkins, J. F., Gesteland, R. F., and Cech, T. R., (Eds.) (2011)    RNA Worlds: From Life's Origins to Diversity in Gene Regulation, 3rd    ed., Cold Spring Harbor Laboratory Press-   2. Cooper, T. A., Wan, L. L., and Dreyfuss, G. (2009) RNA and    Disease, Cell 136, 777-793.-   3. Calin, G. A., and Croce. C. M. (2006) MicroRNAs and chromosomal    abnormalities in cancer cells, Oncogene 25, 6202-6210.-   4. Wilton, S. D., and Fletcher, S. (2005) RNA splicing manipulation:    strategies to modify gene expression for a variety of therapeutic    outcomes, Curr Gene Ther 5, 467-483.-   5. Orr, H. T., and Zoghbi, H. Y. (2007) Trinucleotide repeat    disorders, Annu Rev Neurosci 30, 575-621.-   6. Bates. G. (2003) Huntingtin aggregation and toxicity in    Huntington's disease, Lancet 361, 1642-1644.-   7. Jin, P., Allich, R. S, and Warren, S. T. (2004) RNA and microRNAs    in fragile X mental retardation, Nat Cell Biol 6, 1048-1053.-   8. Sellier, C., Rau, P., Liu, Y., Tassone, F., Hukema, R. K.,    Gattoni, R., Schneider, A., Richard. S., Willemsen. R., Elliott. D.    J., Hagerman, P. J., and Charlet-Berguerand, N. (2010) Sam68    sequestration and partial loss of function are associated with    splicing alterations in FXTAS patients, EMBO J 29, 1248-1261.-   9. Mankodi, A., Logigian, E., Callahan. L., McClain, C., White, R.,    Henderson, D., Krym. M., and Thornton, C. A. (2000) Myotonic    dystrophy in transgenic mice expressing an expanded CUG repeat,    Science 289, 1769-1773.-   10. Kumar, A., Parkesh, R., Sznajder, L J., Childs-Disney, J. L.,    Sobczak, K., and Disney, M. D. (2012) Chemical Correction of    Pre-mRNA Splicing Defects Associated with Sequestration of    Muscleblind-like 1 Protein by Expanded r(CAG)-Containing    Transcripts, ACS Chem Biol. 2012 Mar. 16; 7(3):496-505. Epub 2012    Jan. 17.-   11. Parkesh, R., Childs-Disney, J. L., Nakamori, M., Kumar, A.,    Wang, E., Wang. T., Hoskins, J., Housman, D. E., Thornton, C. A.,    Disney, M. D., and Tran, T. (2012) Design of a Bioactive Small    Molecule that Targets the Myotonic Dystrophy Type 1 RNA Via an RNA    Motif-Ligand Database & Chemical Similarity Searching, J Am Chem    Soc. 2012 Mar. 14; 134(10):4731-42. Epub 2012 Mar. 5.-   12. Childs-Disney, J. L., Hoskins, J., Rzuczek, S., Thornton, C.,    and Disney. M. D. (2012) Rationally Designed Small Molecules    Targeting the RNA that Causes Myotonic Dystrophy Type 1 Are Potently    Bioactive, ACS Chem Biol. 2012 May 18; 7(5):856-62. Epub 2012 Mar.    5.-   13. Cho, J., and Rando, R. R. (2000) Specific binding of Hoechst    33258 to site 1 thymidylate synthase mRNA, Nucleic Acids Res 28,    2158-2163.-   14. Pushechnikov, A., Lee, M. M., Childs-Disney, J. L., Sobczak, K.,    French, J. M., Thornton, C. A., and Disney, M. D. (2009) Rational    design of ligands targeting triplet repeating transcripts that cause    RNA dominant disease: application to myotonic muscular dystrophy    type 1 and spinocerebellar ataxia type 3, J Am Chem Soc 131,    9767-9779.-   15. Warf, M. B., Nakamori, M., Matthys, C. M., Thornton, C. A., and    Berglund. J. A. (2009) Pentamidine reverses the splicing defects    associated with myotonic dystrophy, Proc Natl Acad Sci USA 106,    18551-18556.-   16. Bostrom, J., Greenwood, J. R., and Gottfries, J. (2003)    Assessing the performance of OMEGA with respect to retrieving    bioactive conformations. J Mol Graph Model 21, 449-462.-   17. Grant, J. A., Gallardo, M. A., and Pickup, B. T. (1996) A fast    method of molecular shape comparison. A simple application of a    Gaussian description of molecular shape, J Comput Chem 17,    1653-1666.-   18. Haigh, J. A., Pickup, B. T., Grant, J. A., and    Nicholls, A. (2005) Small molecule shape-fingerprints J Chem Inf    Model 45, 673-684.-   19. Mills, J. B., and Dean, P. M. (1996) Three-dimensional    hydrogen-bond geometry and probability information from a crystal    survey, J Comput Aided Mol Des 10, 607-622.-   20. Sellier, C., Rau, F., Liu, Y. L., Tassone, F., Hukema, R. K.,    Gattoni, R., Schneider. A., Richard, S., Willemsen, R., Elliott, D.    J., Hagerman, P. J., and Charlet-Berguerand, N. (2010) Sam68    sequestration and partial loss of function are associated with    splicing alterations in FXTAS patients, Embo Journal 29, 1248-1261.-   21. Sobczak, K., Michlewski, G., de Mezer, M., Kierzek, E., Krol,    J., Okljniczak, M., Klerzek, R., and Krzyzosiak, W. J. (2010)    Structural diversity of triplet repeat RNAs, J Biol Chem 285,    12755-12764.-   22. Tassone, F., Hagerman, R. J., Loesch, D. Z., Lachiewicz. A.,    Taylor, A. K., and Hagerman. P. J. (2000) Fragile X males with    unmethylated, full mutation trinucleotide repeat expansions have    elevated levels of FMR1 messenger RNA, Am J Med Genet 94, 232-236.-   23. Tassone, F., Hagerman, R. J., Taylor, A. K., and    Hagerman, P. J. (2001) A majority of fragile X males with    methylated, full mutation alleles have significant levels of FMR1    messenger RNA. J Med Genet 38, 453-456.-   24. Willemsen, R., Hoogeveen-Westerveld, M., Reis, S., Holstege, J.,    Severijnen, L A., Nieuwenhuizen, I. M., Schrier, M., van Unen, L.,    Tassone, F., Hoogeveen, A. T., Hagerman, P. J., Mientjes, E. J., and    Oostra, B. A. (2003) The FMR1 CGG repeat mouse displays    ubiquitin-positive intranuclear neuronal inclusions; implications    for the cerebellar tremor/ataxia syndrome, Hum Mol Genet 12,    949-959.-   25. Jin, P., Zarnescu, D. C., Zhang, F., Pearson. C. E.,    Lucchesi, J. C., Moses, K., and Warren, S. T. (2003) RNA-mediated    neurodegeneration caused by the fragile X premutation rCGG repeats    in Drosophila, Neuron 39, 739-747.-   26. Jin, P., Duan, R., Qurashi, A., Qin, Y., Tian. D., Rosser, T.    C., Liu, H., Peng, Y., and Warren, S. T. (2007) Pur alpha binds to    rCGG repeats and modulates repeat-mediated neurodegeneration in a    Drosophila model of fragile X tremor/ataxia syndrome, Neuron 55,    556-564.-   27. Tassone, F., Hagerman, R. J., Garcia-Arocena, D., Khandjian, E.    W., Greco, C. M., and Hagerman, P. J. (2004) Intranuclear inclusions    in neural cells with premumation alleles in fragile X associated    tremor/ataxia syndrome, J Med Genet 41, e43.-   28. Greco, C. M., Berman, R. F., Martin, R. M., Tassone, F.,    Schwartz. P. H., Chang, A., Trapp, B. D., Iwahashi, C., Brunberg,    J., Grigsby, J., Hessl, D., Becker, E. J., Papazian, J., Leebey, M.    A., Hagerman, R. J., and Hagerman, P. J. (2006) Neuropathology of    fragile X-associated tremor/ataxia syndrome (FXTAS), Brain 129,    243-255.-   29. Sellier, C., Hagerman, P., Willemsen, R., and    Charlet-Berguerand, N. DROSHA/DGCR8 sequestration by expanded CGG    repeats leads to global micro-RNA processing alteration in FXTAS    patients [abstract], 12th International Fragile X Conference,    Detroit, Mich.-   30. Chawla, G., Lin, C. H., Han, A., Shiue, L., Ares, M., Jr., and    Black, D. L. (2009) Sam68 regulates a set of alternatively spliced    exons during neurogenesis, Mol Cell Biol 29, 201-213.-   31. Chaires. J. B., Ragazzon, P. A., and Garbett, N. C. (2003) A    competition dialysis assay for the study of structure-selective    ligand binding to nucleic acids, Curr Protoc Nucleic Acid Chem    Chapter 8, Unit 8 3.-   32. Philips, A. V., Timchenko, L. T., and Cooper, T. A. (1998)    Disruption of splicing regulated by a CUG-binding protein in    myotonic dystrophy, Science 280, 737-741.-   33. McLennan, Y., Polussa, J., Tassone, F., and Hagerman, R. (2011)    Fragile x syndrome, Curr Genomics 12, 216-224.-   34. Peyret. N., Seneviratne, P. A., Allawi, H. T., and SantaLucia,    J., Jr. (1999) Nearest-neighbor thermodynamics and NMR of DNA    sequences with internal A.A. C.C, G.G, and T.T mismatches,    Biochemistry 38, 3468-3477.-   35. SantaLucia, J., Jr. (1998) A unified view of polymer, dumbbell,    and oligonucleotide DNA nearest-neighbor thermodynamics, Proc Natl    Acad Sci USA 95, 1460-1465.-   36. Puglisi, J. D., and Tinoco, L, Jr. (1989) Absorbance melting    curves of RNA, Methods Enzymol 180, 304-325.-   37. Disney, M. D., Labuda, L. P., Paul, D. J., Poplawski, S. G.,    Pushechnikov, A., Tran, T., Velagapudi, S. P., Wu, M., and    Childs-Disney. J. L. (2008) Two-dimensional combinatorial screening    identifies specific aminoglycoside-RNA internal loop partners, J Am    Chem Soc 130, 11185-11194.-   38. Tran, T., and Disney, M. D. (2011) Two-dimensional combinatorial    screening of a bacterial rRNA A-site-like motif library: defining    privileged asymmetric internal loops that bind aminoglycosides,    Biochemistry 49, 1833-1842.-   39. Aminova, O., Paul, D. J., Childs-Disney, J. L., and    Disney, M. D. (2008) Two-dimensional combinatorial screening    identifies specific 6′-acylated kanamycin A- and 6′-acylated    neamine-RNA hairpin interactions, Biochemistry 47, 12670-12679.-   40. Tran, T., and Disney, M. D. (2011) Molecular recognition of    6′-N-5-hexynoate kanamycin A and RNA 1×1 internal loops containing    CA mismatches, Biochemistry 50, 962-969.-   41. Childs-Disney, J. L, Wu, M., Pushechnikov, A., Aminova, O., and    Disney, M. D. (2007) A small molecule microarray platform to select    RNA internal loop-ligand interactions, ACS Chem Biol 2, 745-754.-   42. Childs-Disney, J. L, and Disney, M. D. (2008) A simple    ligation-based method to increase the information density in    sequencing reactions used to deconvolute nucleic acid selections,    RNA 14, 390-394.-   43. Paul, D. J., Seedhouse, S. J., and Disney, M. D. (2009)    Two-dimensional combinatorial screening and the RNA Privileged Space    Predictor program efficiently identify aminoglycoside-RNA hairpin    loop interactions, Nucleic Acids Res 37, 5894-5907.-   44. Velagapudi, S. P., Seedhouse, S. J., and Disney, M. D. (2010)    Structure-activity relationships through sequencing (StARTS) defines    optimal and suboptimal RNA motif targets for small molecules, Angew    Chem Int Ed Engl 49, 3816-3818.-   45. Velagapudi, S. P., Seedhouse, S. J., French, J., and    Disney, M. D. (2011) Defining the RNA Internal Loops Preferred by    Benzimidazole Derivatives via 2D Combinatorial Screening and    Computational Analysis, J Am Chem Soc 133, 10111-10118.-   46. Wang, Y., and Rando, R. R. (1995) Specific binding of    aminoglycoside antibiotics to RNA, Chem. Biol, 2, 281-290.-   47. Warf, M. B., and Berglund, J. A. (2007) MBNL binds similar RNA    structures in the CUG repeats of myotonic dystrophy and its pro-mRNA    substrate cardiac troponin T, Rna 13, 2238-2251.-   48. Tran, T., and Disney, M. D. (2010), Biochemistry 49, 1833-1842.

All patents and publications referred to herein are incorporated byreference herein to the same extent as if each individual publicationwas specifically and individually indicated to be incorporated byreference in its entirety.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments and optional features, modification and variation of theconcepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the appended claims.

What is claimed is:
 1. A dimeric r(CGG) binding compound of formula (II)

wherein R¹ is H, (C1-C6)alkyl, or (C1-C6)alkanoyl; R² is H,(C1-C6)alkyl, or (C1-C6)alkanoyl; R³ and R⁴ are independently H,(C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxyalkyl(C1-C6)haloalkoxyalkyl, or (C6-C10)aryl, wherein any alkyl, alkanoyl,alkoxy, or aryl group can be substituted with 0-3 J groups; wherein J isany of halo, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl,hydroxy(C1-C6)alkyl, alkoxy(C1-C6)alkyl, (C1-C6)alkanoyl,(C1-C6)alkanoyloxy, cyano, nitro, azido, R₂N, R₂NC(O), R₂NC(O)O,R₂NC(O)NR, (C1-C6)alkenyl, (C1-C6)alkynyl, (C6-C10)aryl,(C6-C10)aryloxy, (C6-C10)aroyl, (C6-C10)aryl(C1-C6)alkyl,(C6-C10)aryl(C1-C6)alkoxy, (C6-C10)aryloxy(C1-C6)alkyl,(C6-C10)aryloxy(C1-C6)alkoxy, (3- to 9-membered)heterocyclyl, (3- to9-membered)heterocyclyl(C1-C6)alkyl, (3- to9-membered)heterocyclyl(C1-C6)alkoxy, (5- to 10-membered)heteroaryl, (5-to 10-membered)heteroaryl(C1-C6)alkyl, (5- to10-membered)heteroaryl(C1-C6)alkoxy, or (5- to 10-membered)heteroaroyl;R is independently at each occurrence H, (C1-C6)alkyl, or (C6-C10)aryl,wherein any alkyl or aryl group is substituted with 0-3 J; and wherein Lis a linker comprising a polypeptide backbone bonded by two respectivenitrogen atoms thereof to a nitrogen atom of a respective 1,2,3-triazolegroup via a respective (C1-C6)alkylene group optionally furthercomprising a glycyl residue, each respective triazole group being bondedvia a (C1-C6)alkylene group to the respective pyridinium nitrogen atomof each ellipticine scaffold; or a pharmaceutically acceptable saltthereof.
 2. The compound of claim 1 wherein for the dimeric r(CGG)binding compound of formula (II), linker group L is a linker of formula(LI)

wherein n=1, 2, 3, 4, 5, 6, 7, or 8; each independently selected n1=0,1, 2, 3, 4, or 5; and each independently selected n2=1, 2, 3, 4, 5, or6; and wherein a wavy line indicates a position of bonding to therespective pyridinium nitrogen atom of formula (II).
 3. A pharmaceuticalcomposition comprising a compound of claim 1 or 2 and a pharmaceuticallyacceptable excipient.
 4. A method of inhibiting a messenger RNA moleculewith a repeat r(CGG) sequence from binding to a protein with a bindingaffinity for a RNA hairpin loop comprising a non-Watson-Crick G-Gnucleotide pair, comprising contacting the messenger RNA molecule havingthe repeat r(CGG) sequence and an effective amount or concentration of adimeric r(CGG) binding compound of formula (II)

wherein R¹ is H, (C1-C6)alkyl, or (C1-C6)alkanoyl; R² is H,(C1-C6)alkyl, or (C1-C6)alkanoyl; R³ and R⁴ are independently H,(C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxyalkyl(C1-C6)haloalkoxyalkyl, or (C6-C10)aryl, wherein any alkyl, alkanoyl,alkoxy, or aryl group can be substituted with 0-3 J groups; wherein J isany of halo, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl,hydroxy(C1-C6)alkyl, alkoxy(C1-C6)alkyl, (C1-C6)alkanoyl,(C1-C6)alkanoyloxy, cyano, nitro, azido, R₂N, R₂NC(O), R₂NC(O)O,R₂NC(O)NR, (C1-C6)alkenyl, (C1-C6)alkynyl, (C6-C10)aryl,(C6-C10)aryloxy, (C6-C10)aroyl, (C6-C10)aryl(C1-C6)alkyl,(C6-C10)aryl(C1-C6)alkoxy, (C6-C10)aryloxy(C1-C6)alkyl,(C6-C10)aryloxy(C1-C6)alkoxy, (3- to 9-membered)heterocyclyl, (3- to9-membered)heterocyclyl(C1-C6)alkyl, (3- to9-membered)heterocyclyl(C1-C6)alkoxy, (5- to 10-membered)heteroaryl, (5-to 10-membered)heteroaryl(C1-C6)alkyl, (5- to10-membered)heteroaryl(C1-C6)alkoxy, or (5- to 10-membered)heteroaroyl;R is independently at each occurrence H, (C1-C6)alkyl, or (C6-C10)aryl,wherein any alkyl or aryl group is substituted with 0-3 J; and wherein Lis a linker comprising a polypeptide backbone bonded by two respectivenitrogen atoms thereof to a nitrogen atom of a respective 1,2,3-triazolegroup via a respective (C1-C6)alkylene group optionally furthercomprising a glycyl residue, each respective triazole group being bondedvia a (C1-C6)alkylene group to the respective pyridinium nitrogen atomof each ellipticine scaffold; or a pharmaceutically acceptable saltthereof.
 5. The method of claim 4 wherein the repeat r(CGG) sequence isa r(CGG)^(exp) sequence.
 6. The method of claim 4 wherein R¹ is H, orwherein R² is H, or both.
 7. The method of claim 4 wherein R³ and R⁴ areeach methyl.
 8. The method of claim 4 wherein for the dimeric r(CGG)binding compound of formula (II), linker group L is a linker of formula(LI)

wherein p1 =1, 2, 3, 4, 5, 6, 7, or 8; each independently selected p2=0,1, 2, 3, 4, or 5; and each independently selected p3=1, 2, 3, 4, 5, or6; and wherein a wavy line indicates a position of bonding to therespective pyridinium nitrogen atom of the compound of formula (II). 9.The method of claim 8 wherein the compound of formula (II) is of formula2E-nNME

wherein n=1, 2, 3, 4, 5, 6, 7 or 8; or a pharmaceutically acceptablesalt thereof.
 10. The method of claim 9 wherein the contacting is invivo in a patient wherein the inhibiting is medically indicated fortreatment of a condition.
 11. The method of claim 10 wherein the patientis suffering from Fragile X-associated Tremor Ataxia Syndrome.
 12. Amethod of treatment of Fragile X-associated Tremor Ataxia Syndrome,comprising administering to a patient afflicted therewith an effectiveamount or concentration of a dimeric r(CGG) binding compound of formula(II)

wherein R¹ is H, (C1-C6)alkyl, or (C1-C6)alkanoyl; R² is H,(C1-C6)alkyl, or (C1-C6)alkanoyl; R³ and R⁴ are independently H,(C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkoxyalkyl(C1-C6)haloalkoxyalkyl, or (C6-C10)aryl; wherein any alkyl, alkanoyl,alkoxy, or aryl group can be substituted with 0-3 J groups; wherein J isany of halo, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)haloalkyl,hydroxy(C1-C6)alkyl, alkoxy(C1-C6)alkyl, (C1-C6)alkanoyl,(C1-C6)alkanoyloxy, cyano, nitro, azido, R₂N, R₂NC(O), R₂NC(O)O,R₂NC(O)NR, (C1-C6)alkenyl, (C1-C6)alkynyl, (C6-C10)aryl,(C6-C10)aryloxy, (C6-C10)aroyl, (C6-C10)aryl(C1-C6)alkyl,(C6-C10)aryl(C1-C6)alkoxy, (C6-C10)aryloxy(C1-C6)alkyl,(C6-C10)aryloxy(C1-C6)alkoxy, (3- to 9-membered)heterocyclyl, (3- to9-membered)heterocyclyl(C1-C6)alkyl, (3- to9-membered)heterocyclyl(C1-C6)alkoxy, (5- to 10-membered)heteroaryl, (5-to 10-membered)heteroaryl(C1-C6)alkyl, (5- to10-membered)heteroaryl(C1-C6)alkoxy, or (5- to 10-membered)heteroaroyl;R is independently at each occurrence H, (C1-C6)alkyl, or (C6-C10)aryl,wherein any alkyl or aryl group is substituted with 0-3 J; and wherein Lis a linker comprising a polypeptide backbone bonded by two respectivenitrogen atoms thereof to a nitrogen atom of a respective 1,2,3-triazolegroup via a respective (C1-C6)alkylene group optionally furthercomprising a glycyl residue, each respective triazole group being bondedvia a (C1-C6)alkylene group to the respective pyridinium nitrogen atomof each ellipticine scaffold; or a pharmaceutically acceptable saltthereof.
 13. The method of claim 12 wherein R¹ is H.
 14. The method ofclaim 12 wherein R² is H.
 15. The method of claim 12 wherein R³ and R⁴are each methyl.
 16. The method of claim 12 wherein for the dimericr(CGG) binding compound of formula (II), linker group L is a linker offormula (LI)

wherein p1=1, 2, 3, 4, 5, 6, 7, or 8; each independently selected p2=0,1, 2, 3, 4, or 5; and each independently selected p3=1, 2, 3, 4, 5, or6; and wherein a wavy line indicates a position of bonding to therespective pyridinium nitrogen atom of the compound of formula (II). 17.The method of claim 16 wherein the compound of formula (II) is offormula 2E-nNME

wherein n=1, 2, 3, 4, 5, 6, 7 or 8; or a pharmaceutically acceptablesalt thereof.